Casein derived peptides and uses thereof

ABSTRACT

Biologically active peptides that are derived from or are similar to sequences identical with the N-terminus of the αS1 fraction of milk casein. These peptides are capable of stimulating and enhancing immune response, protecting against viral infection, normalizing serum cholesterol levels, and stimulating hematopoiesis. The casein-derived peptides are non-toxic and can be used to treat and prevent immune pathologies, hypercholesterolemia, hematological disorders and viral-related diseases, alone or in combination with other peptides or blood cell stimulating factors.

This is a continuation-in-part of PCT Application No. IL02/00720, filedAug. 29, 2002, which claims the benefit of priority from U.S. patentapplication Ser. No. 09/942,121, filed on Aug. 30, 2001, which is acontinuation-in-part of PCT Application No. IL01/00198, filed Mar. 1,2001, which derives the benefit of priority from Israel PatentApplication No. 134830, filed Mar. 1, 2000. This application is also acontinuation-in-part of U.S. Provisional Patent Application No.60/467,603 filed May 5, 2003. The contents of all of the above listedapplications are hereby incorporated in full by reference.

FIELD OF THE INVENTION

The present invention relates to biologically active peptides that arederived from or are similar to the αS-1 casein of milk. These peptidesare capable of immune modulation and other therapeutic activities,including, but not limited to, stimulating and enhancing immuneresponse, protecting against viral infection, normalizing serumcholesterol levels, and stimulating hematopoiesis. The casein-derivedpeptides of the invention are non-toxic and can be used to treat andprevent immune pathologies, diabetes, hypercholesterolemia,hematological disorders and viral-related diseases.

BACKGROUND OF THE INVENTION Bioactive Molecules from Nutrients

In addition to the nutritional value of many foods, certain fractionsand products of digestive pathways possess the ability to influencephysiological processes. Some of these “extranutritional” constituentsare present in their active form in the whole nutriment, such as theimmunoglobulins in mother's milk and colostrums, phytoestrogens found insoy-based foods, polyphenolic antioxidants from fruits and vitamins.Others are encrypted within nutrient molecules, and are released in anactive form during digestion or food processing, for exampleantihypertensive peptides from lactoglobin [Kitts, D. D. (1999), Can. J.Physiol. Pharmacol. 72:4; 423-434].

Biological Activity in Milk Proteins:

Milk contains a wide variety of proteins that contribute to it's uniquequalities. Some proteins, such as bile-salt stimulated lipase, amylase,beta-casein, lactoferrin, haptocorrin and alpha-antitrypsin assist indigestion and utilization of milk-derived nutrients. Other proteins,such as immunoglobulins, kappa-casein, lyzozyme, lactoferrin andlactalbumin may, in the intact or partially digested form, haveimmunomodulatory and antimicrobial activity. Casein, the predominantmilk protein, has been traditionally defined as composed of threefractions, α, β and γ, according to their electrophoretic mobility [N.J. Hipp, et al. (1952), Dairy Sci., 35:272]. Today casein is definedaccording to the amino acid sequences of each of the subgroups αS1, αS2,β and κ [W. N. Engel et al. (1984), J. Dairy Sci. 67: 1599].

In the course of digestion, the casein proteins are subjected toproteolytic cleavage by acid proteases such as chymosin (rennin),trypsin and pepsin, producing shorter peptides and causing curdling andcalcium sequestration by the resultant protein fragments. A few studieswith milk compounds demonstrated casein-related bacteriocidal activity.U.S. Pat. No. 3,764,670 discloses proteolytic casein digests possessingantibiotic properties against microorganisms. Israel Patent No. 42863describes a casein-derived peptide consisting of 23 amino acids of theN-terminus of casein, possessing anti-bacterial activity. Shimizu et al.describe a short N-terminal fragment derived from αS1 casein peptichydrolyzate having emulsifying properties, suggesting that this might besomehow useful to the food industry (Shimizu, et al. J. of Food Science,1984; 49: 1117-20). The authors investigated the amino acid compositionof the fragment, it's in-vitro emulsifying activity, and noted that itresembled a 23 amino acid long N-terminal fragment of αS-1, concludingthat the fragments were identical. However, no proof of identity wasprovided, and no biological activity was investigated.

In another study, Chabance et al. (Biochimie 1998; 80:155-65) detectedthe presence of casein-derived peptides and peptide fragments in thestomachs and blood of humans after ingestion of yoghurt and milk. Theauthors reported the presence of fragments of bioactive κ-casein(caseinoglycopeptide) and an N-terminal fragment of αS-1 casein havingantibacterial activity, in the blood following digestion. They concludedthat the passage of these peptides, unaltered, into the plasma suggestsa common, transport pathway for their duodenal absorption. No activityof the peptide fragments was demonstrated.

Lahov and Regelson describe a brief (30 minutes) chymosin digest ofwhole, acid-precipitated bovine and human casein, to yield a fractionenriched in an αS-1 casein N-terminal peptide (Lahov and Regelson, FdChem Toxic 1996; 34:131-45), essentially duplicating the teachings ofU.S. Pat. No. 3,764,670 to Katzir-Katchalsky et al. The chymosin digestwas then precipitated with TCA, and characterized by centifugal analysisand short column equilibrium methods. The authors report an N-terminalαS-1 casein peptide fragment, similar to the anti-bacterial “isracidin”reported by Katzir-Katchalsky et al. However, the veracity of theauthor's claims to purification to homogeneity are questionable,considering the repeated detection of mixture of peptides reported indetailed studies of chymosin digest of casein employing sensitiveanalytical techniques (see, for example, Carles et al., FEBS Lett. 1985;115:282-6; McSweeney et al., J. Dairy Res., 1993; 60:401-12, and Yvon,et al. Int. J, Pept. Prot Res, 1989; 34:166-76).

In addition, other physiologically active properties, such as opioid andgrowth factor-like activities have been proposed for casein or itsderivatives [Kitts, D. D., (1999), ibid.].

Immune modulating activity has also been observed in casein peptides.Coste et al. [Coste et al. (1992), Immun. Lett 33: 41-46)] observedenhancement of rat lymphocyte proliferation following treatment with apeptide derived from the C-terminus of p casein. U.S. Pat. Nos.5,506,209, 5,538,952 and 5,707,968, all to Mukerji et al. teach theadministration of human β-casein, recombinant human β-casein, andhydrolysates of both, in a liquid enteral formula, for treatingrespiratory syncytial virus, otitis media, H. influenza and otherinfections in infants. Bovine β-casein was tested, but found to lacksignificant inhibitory activity, leading the authors to conclude that“β-casein from human milk has different bioactivity compared to bovineβ-casein”.

U.S. Pat. Nos. 5,147,853 and 5,344,820 to Dosaka, et al. teach theadministration of a sialic-acid conjugated κ-casein and κ-casein-derivedglyco-macropeptide (GMP) from cow's milk for prevention of bacterial andviral infections in vitro and in vivo in rats. U.S. Pat. No. 5,330,975to Isoda, et al. teaches the use of sialic-acid binding κ-casein andκ-casein peptides for the neutralization of bacterial endotoxins, suchas cholera toxin. Similarly, U.S. Pat. Nos. 5,712,250 to Mukerji, etal., and 5,968,901 to Andersson, et al., teach the use of humanκ-casein, but not bovine κ-casein, for the prevention of bacterial andH. influenza infection. However, these casein compositions taught in theprior art are relatively crude, even following gross fractionation, andnone of these studies have determined the specific sequences in thesecasein peptides which confer their “extranutritional” properties.

Recent studies have detected a correlation between the consumption ofthe A1 β-casein fraction of bovine milk and Ischemic Heart Disease (IHD)in many Western countries (see, for example, M. Laugesen, NZ Med J.2003; 116:U295), leading to development of A1 β-casein-free milk (U.S.Pat. No. 6,570,060 to McLachlan).

Hematopoiesis in Cancer Therapy:

Following high-dose chemotherapy, especially following myeloablativedoses of chemoradiotherapy supported by autologous bone marrow orperipheral blood stem cell transplantation (ASCT) or allogeneic bonemarrow transplantation (BMT), patients are at high risk due topancytopenia. Granulocytopenia may lead to development of serious,occasionally fatal infectious complications from common bacterial,viral, fungal and parasitic agents in the immediate post transplantperiod. Similarly, thrombocytopenia frequently results in bleedingtendency and occasionally, in long lasting platelet dependence. Wheneverresistance to platelets develops, bleeding episodes can be lifethreatening and hemorrhagic complications are frequently lethal. Therisk due to granulocytopenia can be partially overcome by supportivemeasures and most effectively by administration of recombinant humancytokines that can enhance reconstitution of granulocytes, particularlygranulocyte colony stimulation factor (G-CSF) and granulocyte macrophagecolony stimulating factor (GM-CSF). These agents are extremely expensive(approximately $200-400/day/patient) and infrequently cause side effectsdue to hypersensitivity reactions, fever, bone pain and occasionallyvascular leak syndromes, including pericarditis and pleuritis. Some ofthe side effects may be due to other cytokines that may be intrinsicallyreleased by these hematopoietic growth factors. Moreover, the use ofthese hematopoietic growth factors may be prohibitive in patients withtumor cells bearing G-CSF or GM-CSF receptors such as in acute andchronic myeloid leukemias and in myelodysplastic syndromes. Whereasmajor progress in treating patients at risk of pancytopenia has beenachieved from the use of hematopoietic cytokines, no progress has beenmade in the treatment of thrombocytopenia. Following high dosechemotherapy and especially following ASCT, patients are at risk forthrombocytopenia which may last for many months even up to 3 years andsome thromboctyopenic patients may never recover. Many patientspreviously treated with multiple blood products become plateletresistant and hence thrombocytopenia may be impossible to overcome, eventransiently, despite intensive and frequent platelet transfusions from asingle donor. Resistance to platelets and protracted thrombocytopeniarepresent a common cause of death at ASCT centers worldwide.

Currently, several new recombinant cytokines such as recombinant humaninterleukin-3 (rhIL3) and recombinant human interleukin-6 (rhIL6) arebeing investigated as potential agents for enhancingmegakaryocytopoiesis and platelet reconstitution. Unfortunately,preliminary clinical trials showed that although rhIL3 and rhIL6 mayenhance platelet reconstitution, such effects are by no means dramaticand may take considerable time.

Clearly, protracted thrombocytopenia represents a major problem inclinical Bone Marrow Transplant centers today, for which no satisfactorysolution has yet been found.

There is thus a widely recognized need for, and it would be highlyadvantageous to have a safe, inexpensive, rapidly effective andwell-defined stimulator of hematopoiesis, and specificallymegakaryocytopoiesis, devoid of the above limitations.

Thrombopoetin (TPO) in Regulation of Hematopoiesis and PlateletFunction:

TPO appears to be the major regulator of platelet production in vivo,although increase in the kidney- and liver-derived growth factor inplatelet deficiencies is not caused by adaptation of TPO biosynthesis inthese organs. Rather, a “feed-back loop” seems to exist in which thenumber of circulating platelets determines how much of the circulatingTPO is available to the bone marrow for platelet production. Inaddition, it has been demonstrated that TPO is an early acting cytokinewith important multilineage effects: TPO alone, or in combination withother early acting cytokines, can (i) promote viability and suppressapoptosis in progenitor cells; (ii) regulate hematopoietic stem cellproduction and function; (iii) trigger cell division of dormantmultipotent cells; (iv) induce multilineage differentiation and (v)enhance formation of multilineage colonies containing granulocytes,erythrocytes, macrophages, and megakaryocytes (MK, CFU-GEMM). Moreover,TPO stimulates the production of more limited progenitors forgranulocyte/monocyte, megakaryocyte and erythroid colonies, andstimulates adhesion of primitive human bone marrow and megakaryocyticcells to fibronectin and fibrinogen. Thus, TPO is an important cytokinefor clinical hematologists/transplanters: for the mobilization,amplification and ex vivo expansion of stem cells and committedprecursor cells for autologous and allogeneic transplantation [von demBorne, A. E. G. Kr., et al., (1998) Thrombopoietin: it's role inplatelet disorders and as a new drug in clinical medicine. In BailliersClin. Hematol. June:11(2), 427-45].

In addition to TPO effects in hematopoiesis, this potent growth factorprimes platelets for various agonists and modulatesplatelet-extracellular matrix interactions. Although it does not itselfcause platelet aggregation, TPO upregulates ADP-induced aggregation,especially on the second wave of aggregation, upregulates granule (ADP,ATP, serotonin, etc.) release and production of thromboxane B2,increases platelet attachment to collagen and potentiates shear-inducedplatelet aggregation. TPO also stimulates PMN activation, inducing IL-8release and priming oxygen metabolite production, likely enhancingantimicrobial defense.

Clinical studies suggest TPO's value in understanding and treating avariety of hematological conditions. In patients with idiopathicaplastic anemia (AA), elevated TPO levels persist even in remissionfollowing immunosupressive therapy, indicating a hematopoietic defect.TPO is elevated in other forms of aplastic thrombocytopenia as well, butnot in conditions of increased platelet destruction. Apparently, thereactive increase in TPO production is insufficient in cases ofdestructive thrombocytopenia. Thus, TPO is not only a therapeutic optionfor aplastic, but also for destructive thrombocytopenia.

Thrombopoietic agents are of great clinical interest, for preventionand/or treatment of pathological or treatment-induced thrombocytopenia,and as a substitute for platelet transfusions. Of the cytokinesevaluated, all but the marginally potent IL-11 have been deemedunacceptable for clinical use. TPO is widely believed to become thecytokine of choice for throbocytopenia treatment. Recombinant human TPO(Genentech) has recently become available, enabling accuratepharmacokinetic determinations and clinical trials. Thus, TPO'spotential applications encompass the realms of supportive care (postchemo/radio-therapy, bone marrow and stem cell transplantation),hematological disease (AA, myelodysplasia, congenital and acquiredthrombocytopenia), liver diseases, transfusion (expansion, harvest,mobilization and storage of platelets) and surgery (including livertransplantation). Of particular interest is the potential use ofTPO/EPO/G-CSP cocktail for myelodysplasia, G-CSF and TPO combination forperipheral stem cell mobilization and TPO in harvesting CD 34+ cells andex vivo expansion of megakaryocytes for superior plateletreconstitution. Recombinant human G-CSF is also available (Filgrastim,Amgen Inc., USA). However, similar to other hematopoietic agents underconsideration for clinical application, TPO and G-CSF are costly andpotentially antigenic at therapeutically effective levels. Thus, itwould be advantageous to have a safe, inexpensive and readily availablestimulator of thrombopoiesis and granulocytopoiesis capable ofaugmenting TPO and G-CSF activity.

SARS:

The worldwide outbreak of severe acute respiratory syndrome (SARS), andreported SARS-related deaths in more than 25 countries in the spring of2003 has focused attention on the suspected infective agent, theSARS-CoV coronavirus (Rota et al., Sciencexpress, 1 May 2003). Evidenceof SARS-CoV infection has been documented in SARS patients throughoutthe world, SARS-CoV infection has been detected in respiratoryspecimens, and convalescent-phase serum from SARS patients containsanti-SARS antibodies. Presently, no therapies have been identified forthe prevention or treatment of SARS-CoV infection.

In the absence of effective vaccines or drugs, the current SARS epidemicthreatens to reach devastating proportions, similar to epidemics ofother infectious diseases spread by respiratory route such as theinfluenza epidemic of 1918 and measles epidemics. As has beenemphatically stated by many health officials, the key to controllingepidemics is the blockage of transmission of infection. Thus, inaddition to much needed public health measures, the development ofmethods for prevention and/or treatment of SARS is of foremostimportance.

The αS-1, κ-, and β-Fraction of Casein:

The αS1 fraction of casein can be obtained from milk proteins by variousmethods [D. G. Schmidth and T. A. J. Paynes (1963), Biochim., Biophys.Acta, 78:492; M. P. Thompson and C. A. Kiddy (1964), J. Dairy Sci.,47:626; J. C Mercier, et al. (1968), Bull. Soc. Chim. Biol. 50:521], andthe complete amino acid sequence of the αS1 fraction of casein wasdetermined by J. C. Mercier et al. (1971) (Eur. J. Biochem. 23:41). Thegenomic and coding sequences of bovine αS1 fraction of casein have alsobeen cloned and sequenced employing recombinant DNA techniques [D.Koczan, et al. (1991), Nucl. Acids Res. 19(20): 5591; McKnight, R. A.,et al. (1989), J. Dairy Sci. 72:2464-73]. Proteolytic cleavage andidentification of N-terminal fragments from the αS1 fraction of caseinhas been documented [J. C. Mercier, et al. (1970), Eur. J. Biochem.16:439; P. L. H. McSweeney et al. (1993), J. Dairy Res., 60:401], as hasthe intestinal absorption and appearance of this fragment in mammalianplasma following ingestion of whole milk proteins [Fiat, A. M., et al.(1998) Biochimie, 80(2):2155-65]. Meisel, H. and Bockelmann, W. [(1999),Antonie Van Leeuwenhoek, 76:207-15] detected amino acid sequences ofimmunopeptides, casokinins and casomorphins in peptides liberated bylactic acid bacteria digests of α and β casein fractions. Of particularinterest is the anti-aggregating and thrombolytic activity demonstratedfor C-terminal portions of the α- and κ-casein fractions [Chabance, B.et al. (1997), Biochem. Mol. Biol. Int. 42(1) 77-84; Fiat A. M. et al.(1993), J. Dairy Sci. 76(1): 301-310].

The coding sequences for bovine αS2-, β- and κ-casein have also beencloned (Groenen et al., Gene 1993; 123:187-93, Stewart, et al., Mol.Biol Evol. 1987:4:231-41, and Stewart, et al., Nucl. Acids Res. 1984;12:3895-907). The αS2-casein coding sequence has numerous Alu-likeretroposon sequences, and, although the gene is organized similarly tothe αS1-casein gene, sequence analysis indicates that it is more closelyrelated to the β-casein-encoding gene. β-casein is characterized bynumerous clusters of serine residues, which, when phosphorylated, caninteract with and sequester calcium phosphate (Stewart et al., Mol BiolEvol. 1987; 4:231-43). κ-casein is a smaller polypeptide, the amino acidand nucleotide sequence of which (Alexander et al., Eu. J. Biochem.1988; 178:395-401) indicates that it is evolutionarily unrelated to thecalcium-sensitive casein gene family. In the gut, κ-casein is split intoan insoluble peptide (para-kappa casein) and a soluble hydrophilicglycopeptide (caseinomacropeptide), which has been shown to be active inefficiency of digestion, prevention of neonate hypersensitivity toingested proteins, and inhibition of gastric bacterial pathogens(Malkoski, et al., Antimicrob Agents Chemother, 2001; 45:2309-15).

Previous studies have documented potential bioactive peptides encryptedin the N-terminal αS-1 casein, the αS2-casein, β-casein and in theκ-casein amino acid sequences, but no mention was made of use of theseprotein fragments, specific sequences or defined synthetic peptides toenhance hematopoiesis, prevent viral infection or modulate thedevelopment of autoimmune diseases.

The present invention successfully addresses the shortcomings of thepresently known art by providing peptides for the treatment of humandisease, which peptides are derived from the N terminus portion of αS1casein and posses no detectable toxicity and high therapeutic efficacyin a myriad of pathological indications.

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided amethod of preventing or treating an autoimmune or infectious disease orcondition, the method effected by administering to a subject in needthereof a therapeutically effective amount of a peptide derived from theN-terminus portion of αS1 casein.

According to further features in preferred embodiments of the inventiondescribed below the autoimmune or infectious disease or condition isselected from the group consisting of a viral disease, a viralinfection, AIDS, and infection by HIV.

According to another aspect of the present invention there is provided amethod of preventing or treating a blood disease or condition, themethod effected by administering to a subject in need thereof atherapeutically effective amount of a peptide derived from the Nterminus portion of αS1 casein.

According to further features in preferred embodiments of the inventiondescribed below the blood disease or condition is selected from thegroup consisting of thrombocytopenia, pancytopenia, granulocytopenia, anerythropoietin treatable condition, and a thrombopoietin treatablecondition.

According to a yet another aspect of the present invention there isprovided a method of modulating blood cell formation, the methodeffected by administering to a subject in need thereof a therapeuticallyeffective amount of a peptide derived from the N terminus portion of αS1casein.

According to further features in preferred embodiments of the inventiondescribed below the modulating blood cell formation is selected from thegroup consisting of inducing hematopoiesis, inducing hematopoietic stemcells proliferation, inducing hematopoietic stem cells proliferation anddifferentiation, inducing megakaryocytopoiesis, inducing erythropoiesis,inducing leukocytopoiesis, inducing thrombocytopoiesis, inducing plasmacell proliferation, inducing dendritic cell proliferation and inducingmacrophage proliferation.

According to still another aspect of the present invention there isprovided a method of enhancing peripheral stem cell mobilization, themethod effected by administering to a subject in need thereof atherapeutically effective amount of a peptide derived from an N terminusportion of αS1 casein.

According to another aspect of the present invention there is provided amethod of preventing or treating a metabolic disease or condition, themethod effected by administering to a subject in need thereof atherapeutically effective amount of a peptide derived from the Nterminus portion of αS1 casein.

According to further features in preferred embodiments of the inventiondescribed below the metabolic disease or condition is selected from thegroup consisting of NIDDM, IDDM, glucosuria, hyperglycemia,hyperlipidemia, and hypercholesterolemia.

According to another aspect of the present invention there is provided amethod of preventing or treating conditions associated withmyeloablative doses of chemoradiotherapy supported by autologous bonemarrow or peripheral blood stem cell transplantation (ASCT) orallogeneic bone marrow transplantation (BMT), the method effected byadministering to a subject in need thereof a therapeutically effectiveamount of a peptide derived from the N terminus portion of αS1 casein.

According to yet another aspect of the present invention there isprovided a method of augmenting the effect of a blood cell stimulatingfactor, the method effected by administering to a subject in needthereof a therapeutically effective amount of a peptide derived from theN terminus portion of αS1 casein.

According to further features in preferred embodiments of the inventiondescribed below the blood cell stimulating factor is selected from thegroup consisting of thrombopoietin, erythropoietin and granulocytecolony stimulating factor (G-CSF).

According to still another aspect of the present invention there isprovided a method of enhancing colonization of donated blood stem cellsin a myeloablated recipient, the method effected by treating a donor ofthe donated blood stem cells with a therapeutically effective amount ofpeptide derived from the N terminus portion of αS1 casein prior todonation and implanting the donated blood stem cells in the recipient.

According to further features in preferred embodiments of the inventiondescribed below the method further comprising treating the donated bloodcells with a blood cell stimulating factor, the blood cell stimulatingfactor selected from the group consisting of thrombopoietin,erythropoietin and granulocyte colony stimulating factor (G-CSF) priorto implanting the blood stem cells in the recipient.

According to yet another aspect of the present invention there isprovided a method of enhancing colonization of donated blood stem cellsin a myeloablated recipient, the method effected by treating the donatedblood stem cells with a therapeutically effective amount of peptidederived from the N terminus portion of αS1 casein prior to implantingthe donated blood stem cells in the recipient.

According to further features in preferred embodiments of the inventiondescribed below the method further comprising treating the donor with ablood cell stimulating factor, the blood cell stimulating factorselected from the group consisting of thrombopoietin, erythropoietin andgranulocyte colony stimulating factor (G-CSF) prior to donation andimplanting the blood stem cells in the recipient.

According to still another aspect of the present invention there isprovided a method of enhancing colonization of blood stem cells in amyeloablated recipient, the method effected by treating the blood stemcells with a peptide derived from the N terminus portion of αS1 caseinprior to implanting the blood stem cells in the recipient.

According to further features in preferred embodiments of the inventiondescribed below the method further comprising treating the blood stemcells with a blood cell stimulating factor, the blood cell stimulatingfactor selected from the group consisting of thrombopoietin,erythropoietin and granulocyte colony stimulating factor (G-CSF) priorto implanting the blood stem cells in the recipient.

According to another aspect of the present invention there is provided amethod for preventing or treating a condition associated with a SARSinfective agent, the method effected by administering to a subject inneed thereof a therapeutically effective amount of a peptide derivedfrom the N terminus portion of αS1 casein.

According to further features in preferred embodiments of the inventiondescribed below the SARS infective agent is a coronavirus.

According to further features in preferred embodiments of the inventiondescribed below the coronavirus is SARS-CoV.

According to another aspect of the present invention there is provided amethod for preventing or treating a bacterial disease or condition, themethod effected by administering to a subject in need thereof atherapeutically effective amount of a peptide derived from the Nterminus portion of αS1 casein.

According to further features in preferred embodiments of the inventiondescribed below the peptide is a fragment derived from the N terminusportion of αS1 casein by fragmentation of αS1 casein.

According to yet further features in preferred embodiments of theinvention described below the peptide derived from the N terminusportion of αS1 casein is a synthetic peptide.

According to still further features in preferred embodiments of theinvention described below the peptide derived from the N terminusportion of αS1 casein has a sequence as set forth in one of SEQ ID NOs:1-25.

According to further features in preferred embodiments of the inventiondescribed below the peptide derived from the N terminus portion of αS1casein is a peptide derived from natural casein, in combination withpeptides derived from natural non-αS1 casein.

According to further features in preferred embodiments of the inventiondescribed below the method further comprising administering to thesubject in need thereof an effective amount of a blood cell stimulatingfactor, the blood cell stimulating factor selected from the groupconsisting of thrombopoietin, erythropoietin and granulocyte colonystimulating factor (G-CSF).

According to further features in preferred embodiments of the inventiondescribed below the method further comprising administering to thesubject in need thereof an effective amount of erythropoietin,thrombopoietin or granulocyte colony stimulating factor (G-CSF).

According to one aspect of the present invention there is provided apharmaceutical composition for preventing or treating an autoimmune orinfectious disease or condition, the pharmaceutical compositioncomprising, as an active ingredient, a peptide derived from the Nterminus portion of αS1 casein and a pharmaceutically acceptablecarrier.

According to further features in preferred embodiments of the inventiondescribed below the autoimmune or infectious disease or condition isselected from the group consisting of a viral disease, a viralinfection, AIDS, and infection by HIV.

According to another aspect of the present invention there is provided apharmaceutical composition for preventing or treating a blood disease orcondition, the pharmaceutical composition comprising, as an activeingredient, a peptide derived from the N terminus portion of αS1 caseinand a pharmaceutically acceptable carrier.

According to further features in preferred embodiments of the inventiondescribed below the blood disease or condition is selected from thegroup consisting of thrombocytopenia, pancytopenia, granulocytopenia, anerythropoietin treatable condition, and a thrombopoietin treatablecondition and a granulocyte colony stimulating factor treatablecondition.

According to yet another aspect of the present invention there isprovided a pharmaceutical composition for modulating blood cellformation, the pharmaceutical composition comprising, as an activeingredient, a peptide derived from the N terminus portion of αS1 caseinand a pharmaceutically acceptable carrier.

According to further features in preferred embodiments of the inventiondescribed below, modulating blood cell formation is selected from thegroup consisting of inducing hematopoiesis, inducing hematopoietic stemcells proliferation, inducing hematopoietic stem cells proliferation anddifferentiation, inducing megakaryocytopoiesis, inducing erythropoiesis,inducing leukocytopoiesis, inducing thrombocytopoiesis, inducinggranulocytopoiesis, inducing plasma cell proliferation, inducingdendritic cell proliferation and inducing macrophage proliferation.

According to still another aspect of the present invention there isprovided a pharmaceutical composition for enhancing peripheral stem cellmobilization, the pharmaceutical composition comprising, as an activeingredient, a therapeutically effective amount of a peptide derived fromthe N terminus portion of αS1 casein and a pharmaceutically acceptablecarrier.

According to another aspect of the present invention there is provided apharmaceutical composition for preventing or treating a metabolicdisease or condition, the pharmaceutical composition comprising, as anactive ingredient, a peptide derived from the N terminus portion of αS1casein and a pharmaceutically acceptable carrier.

According to further features in preferred embodiments of the inventiondescribed below the metabolic disease or condition is selected from thegroup consisting of NIDDM, IDDM, glucosuria, hyperglycemia,hyperlipidemia, and hypercholesterolemia.

According to yet another aspect of the present invention there isprovided a pharmaceutical composition for preventing or treatingconditions associated with myeloablative doses of chemoradiotherapysupported by autologous bone marrow or peripheral blood stem celltransplantation (ASCT) or allogeneic bone marrow transplantation (BMT),the pharmaceutical composition comprising, as an active ingredient, apeptide derived from the N terminus portion of αS1 casein and apharmaceutically acceptable carrier.

According to still another aspect of the present invention there isprovided a pharmaceutical composition for augmenting the effect of ablood cell stimulating factor, the pharmaceutical compositioncomprising, as an active ingredient, a peptide derived from the Nterminus portion of αS1 casein and a pharmaceutically acceptablecarrier.

According to further features of preferred embodiments in the inventiondescribed below, the blood cell stimulating factor is selected from thegroup consisting of thrombopoietin, erythropoietin and granulocytecolony stimulating factor (G-CSF).

According to another aspect of the present invention there is provided apharmaceutical composition for enhancing colonization of donated bloodstem cells in a myeloablated recipient, the pharmaceutical compositioncomprising, as active ingredients, a peptide derived from the N terminusportion of αS1 casein and a pharmaceutically acceptable carrier.

According to yet another aspect of the present invention there isprovided a pharmaceutical composition for enhancing colonization ofblood stem cells in a myeloablated recipient, the pharmaceuticalcomposition comprising as active ingredients, a peptide derived from theN terminus portion of αS1 casein and a pharmaceutically acceptablecarrier.

According to still another aspect of the present invention there isprovided a pharmaceutical composition for treating or preventing anindication selected from the group consisting of autoimmune disease orcondition, viral disease, viral infection, hematological disease,hematological deficiencies, thrombocytopenia, pancytopenia,granulocytopenia, hyperlipidemia, hypercholesterolemia, glucosuria,hyperglycemia, diabetes, AIDS, HIV-1, helper T-cell disorders, dendritecell deficiencies, macrophage deficiencies, hematopoietic stem celldisorders including platelet, lymphocyte, plasma cell and neutrophildisorders, pre-leukemic conditions, leukemic conditions, immune systemdisorders resulting from chemotherapy or radiation therapy, human immunesystem disorders resulting from treatment of diseases of immunedeficiency and bacterial infections, the pharmaceutical compositioncomprising, as an active ingredient, a peptide derived from the Nterminus portion of αS1 casein and a pharmaceutically acceptablecarrier.

According to another aspect of the present invention there is provided apharmaceutical composition for treating or preventing an indicationselected from the group consisting of hematological disease,hematological deficiencies, thrombocytopenia, pancytopenia,granulocytopenia, dendrite cell deficiencies, macrophage deficiencies,hematopoietic stem cell disorders including platelet, lymphocyte, plasmacell and neutrophil disorders, pre-leukemic conditions, leukemicconditions, myelodysplastic syndrome, non-myeloid malignancies, aplasticanemia and bone marrow insufficiency, the pharmaceutical compositioncomprising, as active ingredients, a blood cell stimulating factor and apeptide derived from the N terminus portion of αS1 casein and apharmaceutically acceptable carrier.

According to one aspect of the present invention there is provided apurified peptide having an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 1-25.

According to another aspect of the present invention there is provided apharmaceutical composition comprising a purified peptide having an aminoacid sequence selected from the group consisting of SEQ ID NOs: 1-25 anda pharmaceutically acceptable carrier.

According to yet another aspect of the present invention there isprovided a pharmaceutical composition comprising a blood cellstimulating factor, said blood cell stimulating factor selected from thegroup consisting of thrombopoietin, erythropoietin and granulocytecolony stimulating factor (G-CSF), in combination with a purifiedpeptide having an amino acid sequence selected from the group consistingof SEQ ID NOs: 1-25 and a pharmaceutically acceptable carrier.

According to still another aspect of the present invention there isprovided a pharmaceutical composition for preventing or treating acondition associated with a SARS infective agent, the pharmaceuticalcomposition comprising, as an active ingredient, a peptide derived fromthe N terminus portion of αS1 casein and a pharmaceutically acceptablecarrier.

According to further features in preferred embodiments of the inventiondescribed below the SARS infective agent is a coronavirus.

According to still further features in preferred embodiments of theinvention described below the coronavirus is SARS-CoV.

According to another aspect of the present invention there is provided apharmaceutical composition for preventing or treating a bacterialinfection the pharmaceutical composition comprising, as an activeingredient, a peptide derived from the N terminus portion of αS1 caseinand a pharmaceutically acceptable carrier.

According to further features in preferred embodiments of the inventiondescribed below the peptide is a fragment derived from the N terminusportion of αS1 casein by fragmentation of αS1 casein.

According to yet further features in preferred embodiments of theinvention described below the peptide derived from the N terminusportion of αS1 casein is a synthetic peptide.

According to still further features in preferred embodiments of theinvention described below the peptide derived from the N terminusportion of αS1 casein has a sequence as set forth in one of SEQ ID NOs:1-25.

According to further features in preferred embodiments of the inventiondescribed below the peptide derived from the N terminus portion of αS1casein is a peptide derived from natural casein, in combination withpeptides derived from natural non-αS1 casein.

According to further features in preferred embodiments of the inventiondescribed below the pharmaceutical composition further comprising, as anactive ingredient, a blood cell stimulating factor, the blood cellstimulating factor selected from the group consisting of thrombopoietin,erythropoietin and granulocyte colony stimulating factor (G-CSF).

According to further features in preferred embodiments of the inventiondescribed below the pharmaceutical composition further comprising, as anactive ingredient, thrombopoietin, erythropoietin or granulocyte colonystimulating factor (G-CSF).

According to still another aspect of the present invention there isprovided a method of low-temperature processing of casein proteolytichydrolysate, the method effected by obtaining a casein proteolytichydrolysate comprising proteolytic enzymes, cooling the caseinproteolytic hydrolysate so as to inactivate the proteolytic enzymes,adjusting the pH of the casein protein hydrolysate to an acid pH,filtering the acidic casein protein hydrolysate, collecting thefiltrate, and further acidifying the filtrate so as to precipitateproteins derived from natural casein, separating and collecting theprecipitate, adjusting the pH of the precipitate to an alkaline pH so asto irreversibly inactivate the proteolytic enzymes; and adjusting the pHof the precipitate to pH 7-9, thereby processing the casein proteinhydrolysate at low temperature.

According to another aspect of the present invention there is provided acasein protein hydrolysate processed at low temperature according to theabovementioned method.

According to further features in preferred embodiments of the inventiondescribed below, step b comprises cooling to about 10° C.

According to still further features in preferred embodiments of theinvention described below adjusting the pH of step c comprises additionof acid to 2% (w/v) acid, and the further acidifying the filtrate ofstep d comprises additional addition of acid to about 10% (w/v) acid.

According to yet further features in preferred embodiments of theinvention described below the alkaline pH of step f is at least pH 9.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIG. 1 depicts the stimulation of Natural Killer (NK) cell activity incultured murine bone marrow cells by peptides derived from naturalcasein. Lysis of ³⁵S labeled YAC target cells by cultured murine bonemarrow cells incubated in the presence or absence of 100 μg per mlpeptides derived from natural casein is expressed as the fraction oftotal radioactivity released from the YAC cells into the culturesupernatant (% Release ³⁵S). FIG. 1 represents NK activity at aneffector:target cell ratio of 25:1 and 50:1.

FIGS. 2 a and 2 b depict the stimulation of Natural Killer (NK) cellactivity in cultured human Peripheral Blood Stem Cells (PBSC) bypeptides derived from natural casein. Lysis of ³⁵S labeled K562 targetcells by cultured human PBSC from Granulocyte Colony Stimulating Factor(G-CSF) treated donors incubated without (0 μg) or with increasingconcentrations (5-500 μg per ml) of peptides derived from natural caseinis expressed as the fraction of total radioactivity released from theK562 cells into the culture supernatant (% Release ³⁵S). FIG. 2 arepresents NK activity of two blood samples from the same patient,incubated at different effector:target cell ratios (100:1 and 50:1).FIG. 2 b represents NK activity of blood samples from normal andaffected donors incubated at a 100:1 effector:target cell ratio. Squaresrepresent an effector:target cell ratio of 100:1, diamonds represent aneffector:target cell ratio of 50:1.

FIGS. 3 a-3 c depict the stimulation of proliferation of Natural Killer(NK) and T-lymphocyte (T) cells from cultured human Peripheral BloodStem Cells (PBSC) by peptides derived from natural casein. NK and T cellproliferation in cultured PBSC from Granulocyte Colony StimulatingFactor treated donors incubated with or without peptides derived fromnatural casein is expressed as the percentage (%) of cells binding theanti-CD₃/FITC fluorescent anti-T cell antibody UCHT₁, or the antiCD₅₆/RPE fluorescent anti-NK cell antibody MOC-1 (DAKO A/S Denmark).Controls are FITC and RPE-conjugated anti-mouse IgG antibody. FIG. 3 arepresents the percentage of cultured human PBSC binding fluorescentantibody CD₅₆ (5 independent samples) after 10 days incubation with(peptides) or without (control) 100 μg per ml peptides derived fromnatural casein. FIG. 3 b represents the percentage of cultured humanPBSC cells binding fluorescent anti-CD₃ (T cell) antibody, following 14days of incubation with (peptides) or without (control) 100 μg per mlpeptides derived from natural casein. FIG. 3 c represents the percentageof cultured human PBSC cells binding fluorescent anti-CD₃ (T cell)antibody and cells binding both CD₃ and CD₅₆ (T and NK-like cells)antibodies after 28 days incubation with (peptides) or without (control)100 μg per ml peptides derived from natural casein.

FIG. 4 depicts the stimulation of Natural Killer (NK) cell activity incultured human Peripheral Blood Stem Cells (PBSC) by synthetic peptidesderived from αS1-casein. Lysis of ³⁵S labeled K562 target cells bycultured human PBSC (from a breast cancer patient) incubated without (0μg) or with increasing concentrations (10-500 μg per ml) of syntheticpeptides derived from casein is expressed as the fraction of totalradioactivity released from the K562 cells into the culture supernatant(% Release). Peptides represent N-terminal sequences of 1-10 (1a,diamonds), 1-11 (2a, squares) and 1-12 (3a, triangles) first amino acidsof the N terminus portion of αS1 casein (see Table 3 below for sequencesof synthetic peptides).

FIGS. 5 a-5 c depict the stimulation of proliferation of cultured humancells of diverse origin by peptides derived from natural casein.Proliferation of the cultured human cells after 14-21 days incubationwith increasing concentrations of the peptides derived from naturalcasein is expressed as the amount of [³H]-thymidine incorporated intoeach sample. FIG. 5 a represents the incorporation of label into twosamples (PBSC 1, squares, 15 days incubation; and PBSC 2, diamonds, 20days incubation) of human Peripheral Blood Stem Cells incubated with orwithout (ctrl) 50-600 μg per ml peptides derived from natural casein.FIG. 5 b represents the incorporation of [³H]-thymidine into culturedhuman bone marrow cells after 21 days incubation with or without (ctrl)50-600 μg per ml peptides derived from natural casein. Bone marrow wasdonated by cancer patients in remission (BM Auto, closed squares, BM 1,triangles, and BM 2,-open squares-) or healthy volunteers (BM normal,diamonds). FIG. 5 c represents incorporation of [³H]-thymidine intocultured human Cord Blood cells after 14 days incubation with or without(ctrl) 50-1000 μg per ml peptides derived from natural casein. Cordblood cells were donated by two separate donors (C.B. 1, triangles, C.B.2, squares).

FIG. 6 shows a Table depicting the proliferation of blood cellprogenitors from human bone marrow and cord blood in response toincubation with peptides derived from natural casein. The relative cellnumber×10⁴ per ml, reflecting the proliferation of cultured cells, wasdetermined by counting cells as described in the Examples section thatfollows. Bone marrow from healthy volunteers (Bone Marrow) and CordBlood from normal births (Cord Blood) was incubated for 13 (Cord Blood)or 14 (Bone Marrow) days in the presence of growth factors and AB serum,with or without increasing concentrations of peptides derived fromnatural casein (25-500 μg/ml).

FIG. 7 shows a table depicting the effect of in-vitro incubation withsynthetic peptides derived from αS1-casein on the relative distributionof Megakaryocyte, Erythroid, Plasma and Dendritic cells (differentialcount) in CFU-GEMM colonies from murine bone marrow progenitor cells.Cells were scored in the macroscopic colonies grown from murine bonemarrow cells prepared similarly to the CFU-GEMM colonies. Cells wereincubated with hematopoietic factors, and 25 μg or more of Syntheticpeptides derived from casein, for 14 days. The differential count isexpressed as the percentage of total cells represented by individualcell types.

FIG. 8 depicts the stimulation of peripheral white blood cellreconstitution in myeloablated, bone marrow transplanted mice inresponse to treatment with peptides derived from natural casein. Cellcounts represent the number of white blood cells (×10⁴ per ml, ascounted in a haemocytometer). The mice (n=6 per group) receivedsub-lethal irradiation and syngeneic bone marrow transplantation (10⁶cells per mouse) on the following day, and intravenous administration of1 mg per recipient peptides derived from natural casein (peptides:squares) or 1 mg per recipient human serum albumin (CONTROL: diamonds)one day later.

FIG. 9 depicts the stimulation of platelet reconstitution inmyeloablated, bone marrow transplanted mice in response to treatmentwith peptides derived from natural casein. Platelet (PLT) countsrepresent the number of thrombocytes (×10⁶ per ml, as counted in ahaemocytometer). The mice (n=7 or 10 per group) received lethalirradiation and syngeneic bone marrow transplantation (10⁶ cells permouse) on day 1, and intravenous administration of 1 mg per recipientpeptides derived from natural casein (Peptides, diamonds) or 1 mg perrecipient human serum albumin (control, squares).

FIGS. 10 a-10 f depict the penetration and nuclear uptake ofFITC-conjugated peptides derived from natural casein in cultured humanT-lymphocyte cells, as recorded by fluorescent microscopy. Sup-T₁ cellswere incubated with 100 μg per ml FITC-conjugated peptides derived fromnatural casein as described in the Examples section that follows. At theindicated times, the cells were washed of free label, fixed in formalinand prepared for viewing and recording by Laser Scanning ConfocalMicroscopy. FIGS. 10 a through 10 f are selected images of cells fromconsecutive incubation times, demonstrating FITC-conjugated peptidesderived from natural casein penetrating the Sup-T₁ cell membrane (FIGS.10 a, 10 b) and concentrating in the nucleus (FIGS. 10 c-10 f).

FIG. 11 shows a Table depicting the stimulation of Sup-T₁ Lymphocytecell proliferation in response to incubation with peptides derived fromnatural casein. Sup-T₁ cells (5000 per well) were incubated withincreasing concentrations (50-1000 μg per ml) of peptides derived fromnatural casein, counted in their wells at the indicated times postculture and pulsed with [³H]-thymidine for 18 hours. Proliferation indexis the ratio of the average of the incorporation of [³H]-thymidine intocells cultured with peptides derived from natural casein (triplicatesamples) divided by the incorporation into cells cultured withoutpeptides derived from natural casein (control).

FIG. 12 shows a Table depicting inhibition of HIV-1 infection of CEMlymphocytes by peptides derived from natural casein. CEM cells wereeither contacted with HIV-1 virus preincubated 3 hours with peptidesderived from natural casein (3 hours), or preincubated themselves withincreasing concentrations (50-1000 μg per ml) of peptides derived fromnatural casein for the indicated number of hours (24 and 48 hours)before contact with HIV-1 virus, as described in the Examples sectionthat follows. On day 15 post infection, cells were counted for cellnumbers and assayed for severity of HIV-1 infection by the P²⁴ antigenassay, as described in the Examples section that follows. Controlcultures were IF: CEM cells contacted with HIV-1 virus withoutpretreatment with peptides derived from natural casein, and UIF: CEMcells cultured under identical conditions without peptides derived fromnatural casein and without contact with HIV-1 virus.

FIG. 13 shows a Table depicting inhibition of HIV-1 infection of CEMlymphocytes by synthetic peptides derived from αS1-casein. CEM cellswere contacted with HIV-1 virus which had been preincubated with variousconcentrations (10-500 μg per ml) of synthetic peptides derived fromαS1-casein (1P, 3P and 4P) for 3 hours (in the presence of thepeptides), as described in the Examples section that follows. On day 7post infection, cells were counted for cell numbers and assayed forseverity of HIV-1 infection by the P²⁴ antigen assay, as described inthe Examples section that follows. Control cultures (IF) were CEM cellscontacted with HIV-1 virus without pretreatment with synthetic peptidesderived from αS1-casein, and UIF: CEM cells cultured under identicalconditions without synthetic peptides derived from casein and withoutcontact with HIV-1 virus.

FIG. 14 depicts the prevention by peptides derived from natural caseinof Type I (IDDM) Diabetes in female Non Obese Diabetic (NOD) mice.Glucosuria was monitored at intervals during 365 days post treatment infemale NOD mice receiving a once (triangles) or twice (squares) weeklyinjection of 100 μg peptides derived from natural casein for 5 weeks (5or 10 injections total) and untreated controls. All the controlsdeveloped glucosuria and subsequently died.

FIG. 15 depicts the reduction by synthetic peptides derived fromαS1-casein of diet-induced hypercholesterol/hyperlipidemia in femaleC57B1/6 mice. Total cholesterol (TC), High Density (HDL) and Low DensityLipoproteins (LDL) were assayed in pooled blood of two (2) mice persample from hypercholesterol/hyperlipidemic mice receiving (IP)casein-derived peptides B, C, 2a or 3P, or no treatment (control).“Normal” samples represent control mice not fed the atherogenic diet.

FIG. 16 shows a Table depicting the stimulation of hematopoiesis incancer patients in response to injections of peptides derived fromnatural casein. Peripheral blood from five female cancer patients eitherreceiving or having received chemotherapy, as described above, wascounted for total White Blood Cells (WBC, ×10³), Platelets (PLT, ×10⁶),Erythrocytes (RBC, ×10³) and Hemoglobin (gm per dl) before (n) and after(n + . . . ) intramuscular injections with peptides derived from naturalcasein. Patient 1 relates to G.T.; patient 2 relates to E.C.; patient 3relates to E.S.; patient 4 relates to J.R. and patient 5 relates to D.M.

FIG. 17 depicts the stimulation by peptides derived from natural caseinof thrombocytopoiesis in a platelet-resistant patient with Acute MyeloidLeukemia (M-1). Thrombocyte reconstitution was expressed as the changein platelet content of peripheral blood (PLT, ×10⁶ per ml), counted asdescribed above at the indicated intervals following intramuscularinjection (as described in the Examples section that follows) of 100 mgpeptides derived from natural casein.

FIG. 18 depicts the stimulation by peptides derived from natural caseinof thrombocytopoiesis in a platelet-resistant patient with Acute MyeloidLeukemia (M-2). Thrombocyte reconstitution was expressed as the changein platelet content of peripheral blood (PLT, ×10⁶ per ml), counted asdescribed above at the indicated intervals following intramuscularinjection (as described in the Examples section that follows) of 100 mgpeptides derived from natural casein.

FIG. 19 shows a table depicting the synergistic effect of incubationwith synthetic peptides derived from αS1-casein on hematopoietic factorstimulation of granulocyte and monocyte colony formation in CFU-GMcolonies from murine bone marrow progenitor cells. Cells were scored inthe macroscopic colonies grown from murine bone marrow cells preparedsimilarly to the CFU-GEMM colonies previously described. Cells wereincubated with hematopoietic factors cytokine (IL-3) and colonystimulating factor (G-CSF), and 25 μg or more of synthetic peptidesderived from casein (J), representing amino acids 1-22 of α-S1 casein,or 30-4, representing amino acids 1-6 of α-S1 casein, for 14 days,individually or in combination. The stimulation of colony formation(CFU) is expressed as the number of myeloid colonies in 10⁵ plated MNCs.Note the synergistic increase in myelocyte formation in cultures exposedto G-CSF, IL-3 and either of the synthetic peptides derived from casein.

FIG. 20 shows a table depicting the synergistic effect of incubationwith synthetic peptides derived from αS1-casein on hematopoietic factorstimulation of granulocyte and monocyte colony formation in CFU-GMcolonies from human bone marrow progenitor cells. Cells were scored inthe macroscopic colonies grown from human bone marrow cells preparedsimilarly to the CFU-GEMM colonies previously described. Cells wereincubated with hematopoietic factors cytokine (IL-3) and colonystimulating factor (G-CSF), and 25 μg or more of synthetic peptidesderived from casein: peptide J, representing amino acids 1-22 of α-S1casein. Exposure of the human bone marrow progenitor cells to peptidesderived from casein was for 14 days. Stimulation of colony formation(CPU) is expressed as the number of myeloid colonies in 10⁵ plated MNCs.Note the synergistic increase (>50% with 100 μg/ml of peptide J) inmyelocyte formation in cultures exposed to G-CSF, IL-3 and the syntheticpeptides derived from an N terminus portion of α-S1 casein.

FIG. 21 shows a table depicting the effect of incubation with syntheticpeptides derived from αS1-casein on Megakaryocytopoiesis in CFU-GEMMcolonies from murine bone marrow progenitor cells. Cells were scored inthe macroscopic colonies grown from murine bone marrow cells preparedsimilarly to the CFU-GEMM colonies previously described. Cells wereincubated with 25 μg or more of synthetic peptides derived from caseinrepresenting amino acids 1-22 of α-S1 casein (J) (SEQ ID NO:21), for 14days. Stimulation of megakaryocyte formation is expressed as the percentof megakaryocytes (differential count). Note the dramatic effect ofsynthetic peptide J representing amino acids 1-22 of α-S1 casein onEarly (E.MK) megakaryocyte formation.

FIG. 22 shows a table depicting the stimulation of plateletreconstitution in myeloablated, bone marrow transplanted mice inresponse to treatment with synthetic peptides derived from αS1-casein[synthetic peptide J, (SEQ ID NO:21) representing amino acids 1-22 ofα-S1 casein]. Cell counts represent the number of platelets (×10³ permm³, as counted in a Coulter Counter). The mice (n=5 per group) receivedsub-lethal irradiation and syngeneic bone marrow transplantation (3×10⁶cells per mouse) on the following day, and intravenous administration of1 mg per recipient of synthetic peptide J (SEQ ID NO: 21) representingamino acids 1-22 of α-S1 casein, or 1 mg per recipient human serumalbumin (CONTROL) one day later. Platelets were measured 10 days later.Note the strong effect (>25% enhancement) of the synthetic peptide J(SEQ ID NO:21) on platelet reconstitution at 10 days post ablation.

FIG. 23 depicts the stimulation of peripheral white blood cellreconstitution in myeloablated, bone marrow transplanted mice inresponse to treatment with peptides derived from natural αS1- andκ-casein. Cell counts represent the mean values of white blood cells(per ml, as counted in a haemocytometer). The mice (n=5 per group)received sub-lethal irradiation and syngeneic bone marrowtransplantation (3×10⁶ cells per mouse) on the following day, andintravenous administration of 1 mg per recipient of α-S1 or κ peptidesderived from natural casein prepared from gel filtration (α-S1 1-23,κ-casein (106-169)), synthetic peptides derived from α-S1 casein (SEQ IDNO:21), or 1 mg per recipient human serum albumin (CONTROL) one daylater. Note the dramatic enhancement of white blood cell reconstitutionby peptides derived from native αS1- and κ casein, and also syntheticpeptides derived from αS1-casein at days 5 and 7 post-reconstitution.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of biologically active peptides that arederived from or are similar to sequences identical with the N-terminusof the αS1-fraction of milk casein, compositions containing same andmethods of utilizing same in, for example, stimulating and enhancingimmune response, protecting against viral infection, normalizing serumcholesterol levels, and stimulating hematopoiesis. The casein-derivedpeptides are non-toxic and can be used to treat and prevent, forexample, immune pathologies, hypercholesterolemia, hematologicaldisorders and viral-related diseases.

The principles and operation of the present invention may be betterunderstood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

As used herein, the term “treating” includes substantially inhibiting,slowing or reversing the progression of a disease, and/or substantiallyameliorating clinical symptoms of a disease.

As used herein, the term “preventing” includes substantially preventingthe appearance of clinical symptoms of a disease.

As used herein the term “peptide” includes native peptides (eitherdegradation products, synthetically synthesized peptides or recombinantpeptides) and peptido-mimetics (typically, synthetically synthesizedpeptides), such as peptoids and semipeptoids which are peptide analogs,which may have, for example, modifications rendering the peptides morestable while in a body. Such modifications include, but are not limitedto, cyclization, N terminus modification, C terminus modification,peptide bond modification, including, but not limited to, CH₂—NH, CH₂—S,CH₂—S═O, O═C—NH, CH₂—O, CH₂—CH₂, S═C—NH, CH═CH or CF═CH, backbonemodification and residue modification. Methods for preparingpeptido-mimetic compounds are well known in the art and are specified,for example, in Quantitative Drug Design, C. A. Ramsden Gd., Chapter17.2, F. Choplin Pergamon Press (1992), which is incorporated byreference as if fully set forth herein. Further detail in this respectare provided hereinunder.

Thus, a peptide according to the present invention can be a cyclicpeptide. Cyclization can be obtained, for example, through amide bondformation, e.g., by incorporating Glu, Asp, Lys, Orn, di-amino butyric(Dab) acid, di-aminopropionic (Dap) acid at various positions in thechain (—CO—NH or —NH—CO bonds). Backbone to backbone cyclization canalso be obtained through incorporation of modified amino acids of theformulas H—N((CH₂)_(n)—COOH)—C(R)H—COOH orH—N((CH₂)_(n)—COOH)—C(R)H—NH₂, wherein n=1-4, and further wherein R isany natural or non-natural side chain of an amino acid.

Cyclization via formation of S—S bonds through incorporation of two Cysresidues is also possible. Additional side-chain to side chaincyclization can be obtained via formation of an interaction bond of theformula —(—CH₂—)_(n)—S—CH₂—C—, wherein n=1 or 2, which is possible, forexample, through incorporation of Cys or homoCys and reaction of itsfree SH group with, e.g., bromoacetylated Lys, Orn, Dab or Dap.

Peptide bonds (—CO—NH—) within the peptide may be substituted, forexample, by N-methylated bonds (—N(CH₃)—CO—), ester bonds(—C(R)H—C—O—O—C(R)—N—), ketomethylen bonds (—CO—CH₂—), α-aza bonds(—NH—N(R)—CO—), wherein R is any alkyl, e.g., methyl, carba bonds(—CH₂—NH—), hydroxyethylene bonds (—CH(OH)—CH₂—), thioamide bonds(—CS—NH—), olefinic double bonds (—CH═CH—), retro amide bonds (—NH—CO—),peptide derivatives (—N(R)—CH₂—CO—), wherein R is the “normal” sidechain, naturally presented on the carbon atom.

These modifications can occur at any of the bonds along the peptidechain and even at several (2-3) at the same time.

Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted forsynthetic non-natural acid such as TIC, naphthylelanine (Nol),ring-methylated derivatives of Phe, halogenated derivatives of Phe oro-methyl-Tyr.

Tables 1-2 below list all the naturally occurring amino acids (Table 1)and non-conventional or modified amino acids (Table 2).

TABLE 1 Three-Letter Amino Acid Abbreviation One-letter Symbol AlanineAla A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys CGlutamine Gln Q Glutamic Acid Glu E Glycine Gly G Histidine His HIsoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met MPhenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr TTryptophan Trp W Tyrosine Tyr Y Valine Val V Any amino acid as above XaaX

TABLE 2 Non-conventional amino acid Code Non-conventional omino acidCode α-aminobutyric acid Abu L-N-methylalanine Nmalaα-amino-α-methylbutyrate Mgabu L-N-methylarginine Nmargaminocyclopropane- Cpro L-N-methylasparagine Nmasn CarboxylateL-N-methylaspartic acid Nmasp aminoisobutyric acid AibL-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmgincarboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine ChexaL-N-methylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucineNmile D-alanine Dal L-N-methylleucine Nmleu D-arginine DargL-N-methyllysine Nmlys D-aspartic acid Dasp L-N-methylmethionine NmmetD-cysteine Dcys L-N-methylnorleucine Nmnle D-glutamine DglnL-N-methylnorvaline Nmnva D-glutamic acid Dglu L-N-methylornithine NmornD-histidine Dhis L-N-methylphenylalanine Nmphe D-isoleucine DileL-N-methylproline Nmpro D-leucine Dleu L-N-methylserine Nmser D-lysineDlys L-N-methylthreonine Nmthr D-methionine Dmet L-N-methyltryptophanNmtrp D-ornithine Dorn L-N-methyltyrosine Nmtyr D-phenylalanine DpheL-N-methylvaline Nmval D-proline Dpro L-N-methylethylglycine NmetgD-serine Dser L-N-methyl-t-butylglycine Nmtbug D-threonine DthrL-norleucine Nle D-tryptophan Dtrp L-norvaline Nva D-tyrosine Dtyrα-methyl-aminoisobutyrate Maib D-valine Dval α-methyl-γ-aminobutyrateMgabu D-α-methylalanine Dmala α-methylcyclohexylalanine MchexaD-α-methylarginine Dmarg α-methylcyclopentylalanine McpanD-α-methylasparagine Dmasn α-methyl-α-napthylalanine ManapD-α-methylaspartate Dmasp α-methylpenicillamine Mpen D-α-methylcysteineDmeys N-(4-aminobutyl)glycine Nglu D-α-methylglutamine DmglnN-(2-aminoethyl)glycine Naeg D-α-methylhistidine DmhisN-(3-aminopropyl)glycine Norn D-α-methylisoleucine DmileN-amino-α-methylbutyrate Nmaabu D-α-methylleucine Dmleu α-napthylalanineAnap D-α-methyllysine Dmlys N-benzylglycine Nphe D-α-methylmethionineDmmet N-(2-carbamylethyl)glycine Ngln D-α-methylornithine DmornN-(carbamylmethyl)glycine Nasn D-α-methylphenylalanine DmpheN-(2-carboxyethyl)glycine Nglu D-α-methylproline DmproN-(carboxymethyl)glycine Nasp D-α-methylserine Dmser N-cyclobutylglycineNcbut D-α-methylthreonine Dmthr N-cycloheptylglycine NchepD-α-methyltryptophan Dmtrp N-cyclohexylglycine Nchex D-α-methyltyrosineDmty N-cyclodecylglycine Ncdec D-α-methylvaline DmvalN-cyclododeclglycine Ncdod D-α-methylalnine Dnmala N-cyclooctylglycineNcoct D-α-methylarginine Dnmarg N-cyclopropylglycine NcproD-α-methylasparagine Dnmasn N-cycloundecylglycine NcundD-α-methylasparatate Dnmasp N-(2,2-diphenylethyl)glycine NbhmD-α-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine NbheD-N-methylleucine Dnmleu N-(3-indolylyethyl) glycine NhtrpD-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate NmgabuN-methylcyclohexylalanine Nmchexa D-N-methylmethionine DnmmetD-N-methylornithine Dnmorn N-methylcyclopentylalanine NmcpenN-methylglycine Nala D-N-methylphenylalanine DnmpheN-methylaminoisobutyrate Nmaib D-N-methylproline DnmproN-(1-methylpropyl)glyclne Nile D-N-methylserine DnmserN-(2-methylpropyl)glycine Nile D-N-methylserine DnmserN-(2-methylpropyl)glycine Nleu D-N-methylthreonine DnmthrD-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine NvaD-N-methyltyrosine Dnmtyr N-methyla-napthylalanine NmanapD-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acidGabu N-(p-hydroxyphenyl)glycine Nhtyr L-t-butylglycine TbugN-(thiomethyl)glycine Ncys L-ethylglycine Etg Penicillamine PenL-homophenylalanine Hphe L-α-methylalanine Mala L-α-methylarginine MargL-α-methylasparagine Masn L-α-methylaspartate MaspL-α-methyl-t-butylglycine Mtbug L-α-methylcysteine McysL-melhylethylglycine Metg L-α-methylglutamine Mgln L-α-methylglutamateMglu L-α-methylhistidine Mhis L-α-methylhomo phenylalanine MhpheL-α-methylisoleucine Mile N-(2-methylthioethyl)glycine NmetD-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine NargD-N-methylglutamate Dnmglu N-(1-hydroxyethyl)glycine NthrD-N-methylhistidine Dnmhis N-(hydroxyethyl)glycine NserD-N-methylisoleucine Dnmile N-(imidazolylethyl)glycine NhisD-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine NhtrpD-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate NmgabuN-methylcyclohexylalanine Nmchexa D-N-methylmethionine DnmmetD-N-methylorinithine Dnmorn N-methylcyclopentylalanine NmcpenN-methylglycine Nala D-N-methylphenylalanine DnmpheN-methylaminoisobutyrate Nmaib D-N-methylproline DnmproN-(1-methylpropyl)glycine Nile D-N-methylserine DnmserN-(2-methylpropyl)glycine Nleu D-N-methylthreonine DnmthrD-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine NvalD-N-methyltyrosine Dnmtyr N-methyla-napthylalanine NmanapD-N-methylvaline Dnmval N-methytpenicillamine Nmpen γ-aminobutyric acidGabu N-(p-hydroxyphenyl)glycine Nhtyr L-t-butylglycine TbugN-(thiomethyl)glycine Ncys L-ethylglycine Etg Penicillamine PenL-homophenylalanine Hphe L-α-methylalanine Mala L-α-methylarginine MargL-α-methylasparagine Masn L-α-methylasparate MaspL-α-methyl-t-butylglycine Mtbug L-α-methylcysteine McysL-methylethylglycine Metg L-α-methylglutamine Mgln L-α-methylglutamateMglu L-α-methylhistidine Mhis L-α-methylhomophenylalanine MhpheL-α-methylisoleucine Mile N-(2-methylthioethyl)glycine NmetL-α-methylleucine Mleu L-α-methyllysine Mlys L-α-methylmethionine MmetL-α-methylnorleucine Mnle L-α-methylnorvaline Mnva L-α-methylornithineMorn L-α-methylphenylalanine Mphe L-α-methylproline MproL-α-methylserine Mser L-α-methylthreonine Mthr L-α-methylvaline MtrpL-α-methyltyrosine Mtyr L-α-methylleucine MvalL-N-methylhomophenylalanine Nmhphe Nnbhm N-(N-(2,2-diphenylethyl)N-(N-(3,3-diphenylpropyl) carbamylmethyl-glycine Nnbhmcarbamylmethyl(1)glycine Nnbhe 1-carboxy-1-(2,2-diphenyl Nmbcethylamino)cyclopropane

A peptide according to the present invention can be used in a selfstanding form or be a part of moieties such as proteins and displaymoieties such as display bacteria and phages. The peptides of theinvention can also be chemically modified to give active dimers ormultimers, in one polypeptide chain or covalently crosslinked chains.

Additionally, a peptide according to the present invention includes atleast two, optionally at least three, optionally at least four,optionally at least five, optionally at least six, optionally at leastseven, optionally at least eight, optionally at least nine, optionallyat least ten, optionally at least eleven, optionally at least twelve,optionally at least thirteen, optionally at least fourteen, optionallyat least fifteen, optionally at least sixteen, optionally at leastseventeen, optionally at least eighteen, optionally at least nineteen,optionally at least twenty, optionally at least twenty-one, optionallyat least twenty-two, optionally at least twenty-three, optionally atleast twenty-four, optionally at least twenty-five, optionally at leasttwenty-six, optionally between twenty-seven and sixty, or more aminoacid residues (also referred to herein interchangeably as amino acids).

Accordingly, as used herein the term “amino acid” or “amino acids” isunderstood to include the 20 naturally occurring amino acids; thoseamino acids often modified post-translationally in vivo, including, forexample, hydroxyproline, phosphoserine and phosphothreonine; and otherunusual amino acids including, but not limited to, 2-aminoadipic acid,hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine,Furthermore, the term “amino acid” includes both D- and L-amino acids.

As used herein the phrase “derived from an N terminus portion ofαS1-casein” refers to peptides as this term is defined herein, e.g.,cleavage products of αS1-casein (referred to herein as peptides derivedfrom natural casein), synthetic peptides chemically synthesized tocorrespond to the amino acid sequence of αS1-casein (referred to hereinas synthetic peptides derived from casein), peptides similar(homologous) to αS1-casein, for example, peptides characterized by oneor more amino acid substitutions, such as, but not limited to,permissible substitutions, provided that at least 70%, preferably atleast 80%, more preferably at least 90% similarity is maintained, andfunctional homologues thereof. The terms “homologues” and “functionalhomologues” as used herein mean peptides with any insertions, deletionsand substitutions which do not affect the biological activity of thepeptide.

As used herein, the phrase “peptides derived from an N terminus portionof αS1-, and combinations thereof” also refers to the abovementionedpeptides in combination with one another. As used herein, the phrase“combination thereof” is defined as any of the abovementioned peptides,derived from αS1-casein, combined in a mixture with one or moreadditional, non-identical or identical peptides derived from αS1-casein.As used herein, the term “mixture” is defined as a non-covalentcombination of peptides existing in variable proportions to one another

The peptides of the present invention may be produced by recombinantmeans or may be chemically synthesised by, for example, the stepwiseaddition of one or more amino acid residues in defined order using solidphase peptide synthetic techniques. Where the peptides may need to besynthesised in combination with other proteins and then subsequentlyisolated by chemical cleavage or alternatively the peptides orpolyvalent peptides may be synthesised in multiple repeat units. Thepeptides may comprise naturally occurring amino acid residues or mayalso contain non-naturally occurring amino acid residues such as certainD-isomers or chemically modified naturally occurring residues. Theselatter residues may be required, for example, to facilitate or provideconformational constraints and/or limitations to the peptides. Theselection of a method of producing the subject peptides will depend onfactors such as the required type, quantity and purity of the peptidesas well as ease of production and convenience.

The peptides of the present invention may first require their chemicalmodification for use in vivo. Chemical modification of the subjectpeptides may be important to improve their biological activity. Suchchemically modified peptides are referred to herein as “analogues”. Theterm “analogues” extends to any functional chemical or recombinantequivalent of the peptides of the present invention, characterised, in amost preferred embodiment, by their possession of at least one of theabovementioned biological activities. The term “analogue” is also usedherein to extend to any amino acid derivative of the peptides asdescribed above.

Analogues of the peptides contemplated herein include, but are notlimited to, modifications to side chains, incorporation of unnaturalamino acids and/or their derivatives during peptide synthesis and theuse of crosslinkers and other methods which impose conformationalconstraints on the peptides or their analogues.

Examples of side chain modifications contemplated by the presentinvention include modifications of amino groups such as by reductivealkylation by reaction with an aldehyde followed by reduction withNaBH₄, amidination with methylacetimidate; acylation with aceticanhydride; carbamoylation of amino groups with cyanate;trinitrobenzylation of amino groups with 2,4,6-trinitrobenzene sulphonicacid (TNBS); acylation of amino groups with succinic anhydride andtetrahydrophthalic anhydride; and pyridoxylation of lysine withpyridoxal-5′-phosphate followed by reduction with NaBH₄.

The guanidine group of arginine residues may be modified by theformation of heterocyclic condensation products with reagents such as2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation viaO-acylisourea formation followed by subsequent derivitisation, forexample, to a corresponding amide.

Sulphydryl groups may be modified by methods such as carboxymethylationwith iodoacetic acid or iodoacetamide; performic acid oxidation tocysteic acid; formation of a mixed disulphides with other thiolcompounds; reaction with maleimide, maleic anhydride or othersubstituted maleimide; formation of mercurial derivatives using4-chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid,phenylmercury chloride, 2-chloromercuri-4-nitrophenol and othermercurials; carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation withN-bromosuccinimide or alkylation of the indole ring with2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residueson the other hand, may be altered by nitration with tetranitromethane toform a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may beaccomplished by alkylation with iodoacetic acid derivatives orN-carbethoxylation with diethylpyrocarbonate.

Examples of incorporating unnatural amino acids and derivatives duringpeptide synthesis include, but are not limited to, use of norleucine,4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid,6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine,ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid,2-thienyl alanine and/or D-isomers of amino acids.

As used herein the terms “αS1 casein” refers to αS1 casein of a mammal,including, but not limited to, livestock mammals (e.g., cow, sheep,goat, mare, camel, deer and buffalo) human beings and marine mammals.The following provides a list of αS1 caseins having a known amino acidsequence, identified by their GenBank (NCBI) Accession Nos. and source:αS1 caseins: CAA26982 (Ovis aries (sheep)), CAA51022 (Capra hircus(goat)), CAA42516 (Bos taurus (bovine)), CAA55I85 (Homo sapiens),CAA38717 (Sus scrofa (pig)), P09115 (rabbit) and O97943 (Camelusdromedurius (camel)).

As used herein the term “N terminus portion” refers to M amino acids ofαS1 casein derived from the first 60 amino acids of αS1 casein, whereinM is any of the integers between 2 and 60 (including the integers 2 and60). Preferably, the term refers to the first M amino acids of αS1casein.

The peptides of the invention can be obtained by extraction from milk aspreviously described, or by solid phase peptide synthesis, which is astandard method known to the man skilled in the art. Purification of thepeptides of the invention is performed by standard techniques, known toone skilled in the art, such as high performance liquid chromatography(HPLC), diafiltration on rigid cellulose membranes (Millipore) and gelfiltration. Milk casein fragmentation to obtain the peptides of theinvention may be effected using various enzymatic and/or chemical means,as described hereinbelow.

As is further detailed hereinunder and exemplified in the Examplessection that follows, the peptides of the present invention have avariety of therapeutic effects. In the Examples section there areprovided numerous assays with which one of ordinary skills in the artcan test a specific peptide designed in accordance with the teachings ofthe present invention for a specific therapeutic effect Any of thepeptides described herein can be administered per se or be formulatedinto a pharmaceutical composition which can be used for treating orpreventing a disease. Such a composition includes as an activeingredient any of the peptides described herein and a pharmaceuticallyacceptable carrier.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the peptides described herein, with other chemicalcomponents such as pharmaceutically suitable carriers and excipients.The purpose of a pharmaceutical composition is to facilitateadministration of a compound to an organism.

Hereinafter, the term “pharmaceutically acceptable carrier” refers to acarrier or a diluent that does not cause significant irritation to anorganism and does not abrogate the biological activity and properties ofthe administered compound. Examples, without limitations, of carriersare: propylene glycol, saline, emulsions and mixtures of organicsolvents with water. Herein the term “excipient” refers to an inertsubstance added to a pharmaceutical composition to further facilitateadministration of a compound. Examples, without limitation, ofexcipients include calcium carbonate, calcium phosphate, various sugarsand types of starch, cellulose derivatives, gelatin, vegetable oils andpolyethylene glycols.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences” Mack Publishing Co., Easton, Pa.,latest edition.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, transdermal, intestinal or parenteral delivery,including intramuscular, subcutaneous and intramedullary injections aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore pharmaceutically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active peptides intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the peptides of the invention may be formulated inaqueous solutions, preferably in physiologically compatible buffers suchas Hank's solution, Ringer's solution, or physiological saline bufferwith or without organic solvents such as propylene glycol, polyethyleneglycol. For transmucosal administration, penetrants are used in theformulation. Such penetrants are generally known in the art.

For oral administration, the peptides can be formulated readily bycombining the active peptides with pharmaceutically acceptable carrierswell known in the art. Such carriers enable the peptides of theinvention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions, and the like, for oralingestion by a patient Pharmacological preparations for oral use can bemade using a solid excipient, optionally grinding the resulting mixture,and processing the mixture of granules, after adding suitableauxiliaries if desired, to obtain tablets or dragee cores. Suitableexcipients are, in particular, fillers such as sugars, includinglactose, sucrose, mannitol, or sorbitol; cellulose preparations such as,for example, maize starch, wheat starch, rice starch, potato starch,gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/orphysiologically acceptable polymers such as polyvinylpyrrolidone (PVP).If desired, disintegrating agents may be added, such as cross-linkedpolyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such assodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active ingredient doses.

Pharmaceutical compositions, which can be used orally, include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theactive peptides may be dissolved or suspended in suitable liquids, suchas fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the peptides according to the presentinvention are conveniently delivered in the form of an aerosol spraypresentation from a pressurized pack or a nebulizer with the use of asuitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. Inthe case of a pressurized aerosol, the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, e.g., gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

The peptides described herein may be formulated for parenteraladministration, e.g., by bolus injection or continuous infusion.Formulations for injection may be presented in unit dosage form, e.g.,in ampoules or in multidose containers with optionally, an addedpreservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-soluble form.Additionally, suspensions of the active peptides may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidsesters such as ethyl oleate, triglycerides or liposomes. Aqueousinjection suspensions may contain substances, which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol or dextran. Optionally, the suspension may also containsuitable stabilizers or agents that increase the solubility of thepeptides to allow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free water,before use.

The peptides of the present invention may also be formulated in rectalcompositions such as suppositories or retention enemas, using, e.g.,conventional suppository bases such as cocoa butter or other glycerides.

The pharmaceutical compositions herein described may also comprisesuitable solid of gel phase carriers or excipients. Examples of suchcarriers or excipients include, but are not limited to, calciumcarbonate, calcium phosphate, various sugars, starches, cellulosederivatives, gelatin and polymers such as polyethylene glycols.

Persons ordinarily skilled in the art can easily determine optimumdosages and dosing methodology for any of the peptides of the invention.

For any peptide used in accordance with the teachings of the presentinvention, a therapeutically effective amount, also referred to as atherapeutically effective dose, which can be estimated initially fromcell culture assays or in vivo animal assays. For example, a dose can beformulated in animal models to achieve a circulating concentration rangethat includes the IC₅₀ or the IC₁₀₀ as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Initial dosages can also be estimated from in vivo data. Usingthese initial guidelines one having ordinary skill in the art coulddetermine an effective dosage in humans.

Moreover, toxicity and therapeutic efficacy of the peptides describedherein can be determined by standard pharmaceutical procedures in cellcultures or experimental animals, e.g., by determining the LD₅₀ and theED₅₀. The dose ratio between toxic and therapeutic effect is thetherapeutic index and can be expressed as the ratio between LD₅₀ andED₅₀. Peptides which exhibit high therapeutic indices are preferred. Thedata obtained from these cell cultures assays and animal studies can beused in formulating a dosage range that is not toxic for use in human.The dosage of such peptides lies preferably within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. The exactformulation, route of administration and dosage can be chosen by theindividual physician in view of the patient's condition (see, e.g.,Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics,chapter 1, page 1).

Dosage amount and interval may be adjusted individually to provideplasma levels of the active ingredient which are sufficient to maintaintherapeutic effect. Usual patient dosages for oral administration rangefrom about 1-1000 mg/kg/administration, commonly from about 10-500mg/kg/administration, preferably from about 20-300 mg/kg/administrationand most preferably from about 50-200 mg/kg/administration. In somecases, therapeutically effective serum levels will be achieved byadministering multiple doses each day. In cases of local administrationor selective uptake, the effective local concentration of the drug maynot be related to plasma concentration. One having skill in the art willbe able to optimize therapeutically effective local dosages withoutundue experimentation.

Depending on the severity and responsiveness of the condition to betreated, dosing can also be a single administration of a slow releasecomposition, with course of treatment lasting from several days toseveral weeks or until cure is effected or diminution of the diseasestate is achieved.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as FDA approved kit, which may containone or more unit dosage forms containing the active ingredient. The packmay, for example, comprise metal or plastic foil, such as a blisterpack. The pack or dispenser device may be accompanied by instructionsfor administration. The pack or dispenser may also be accompanied by anotice associated with the container in a form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals, which notice is reflective of approval by the agency ofthe form of the compositions or human or veterinary administration. Suchnotice, for example, may be of labeling approved by the U.S. Food andDrug Administration for prescription drugs or of an approved productinsert. Compositions comprising a peptide of the invention formulated ina compatible pharmaceutical carrier may also be prepared, placed in anappropriate container, and labeled for treatment or prevention of anindicated condition or induction of a desired event Suitable indica onthe label may include treatment and/or prevention of an autoimmunedisease or condition, viral disease, viral infection, bacterialinfection, hematological disease, hematological deficiencies,thrombocytopenia, pancytopenia, granulocytopenia, an erythropoietintreatable condition, a thrombopoietin treatable condition,hyperlipidemia, hypercholesterolemia, glucosuria, hyperglycemia,diabetes, AIDS, infection with HIV-1, a coronavirus or SARS infection,helper T-cell disorders, dendrite cell deficiencies, macrophagedeficiencies, hematopoietic stem cell disorders including platelet,lymphocyte, plasma cell and neutrophil disorders, hematopoietic stemcell proliferation, hematopoietic stem cell proliferation anddifferentiation, pre-leukemic conditions, leukemic conditions, immunesystem disorders resulting from chemotherapy or radiation therapy, andhuman immune system disorders resulting from treatment of diseases ofimmune deficiency.

The pharmaceutical compositions according to the invention may be usefulin maintaining and/or restoring blood system constituents, in balancingblood cell counts, in balancing levels of metabolites in the bloodincluding sugar, cholesterol, calcium, uric acid, urea and enzymes suchas alkaline phosphatase. Further, the pharmaceutical compositions of theinvention may be useful in inducing blood cell proliferation, modulatingwhite and/or red blood cell counts, particularly increasing white and/orred blood cell counts, elevating haemoglobin blood level and inmodulating platelet counts.

The term “balancing” as used herein with relation to levels of certainphysiological parameters, means changing the levels of referredparameters and bringing them closer to normal values.

As used herein, the term “modulating”, with regard to physiologicalprocesses such as blood cell formation, is defined as effecting a changein the quality and/or amount of said processes, including, but notlimited to, increasing and decreasing frequency, character, duration,outcome, magnitude, cyclic nature, and the like. Examples of suchmodulation are αS1 casein's enhancement of megakaryocyte proliferation,dendritic cell proliferation, and effect of G-CSF on CFU-GM colonygrowth, as described hereinbelow.

The term “normal values” as used herein with relation to physiologicalparameters, means values which are in the range of values of healthyhumans or animals.

In specifically preferred embodiments, the peptides of the invention areuse to treat or prevent blood disease or conditions, and balance countsof red blood cells, white blood cells, platelets and haemoglobin level.The pharmaceutical compositions of the invention may be used formodulating blood cell formation, including activating blood cellproliferation.

In addition, the pharmaceutical compositions may be used for thetreatment and/or prevention of hemopoietic stem cell disorders,including erythrocyte, platelet, lymphocyte, plasma cell, dendriticcell, macrophage and neutrophil disorders, as well as deficiency andmalfunction in pre-leukemic and leukemic conditions andthrombocytopenia.

Further, the pharmaceutical compositions may be used for the treatmentand/or prevention of cell proliferative diseases. In this connection, itis worth noting that the pharmaceutical compositions of the inventionare advantageous in the stimulation of the immune response duringchemotherapy or radiation treatments, in alleviating the negativeeffects, reducing chemotherapy and irradiation-induced vomiting andpromoting a faster recovery.

Still further, the pharmaceutical compositions of the invention may beused for the stimulation of human immune response during treatment ofdiseases associated with immune deficiency, for example HIV andautoimmune diseases.

The compositions of the invention may also be intended for veterinaryuse.

The pharmaceutical compositions of the invention may be used in thetreatment and/or prevention of, for example, disorders involvingabnormal levels of blood cells, disorders involving hemopoietic stemcells production and differentiation, treatment of platelet, lymphocyte,dendritic cell and/or neutrophil disorders, for the treatment ofpre-leukemic and leukemic conditions and for the treatment ofthrombocytopenia. The pharmaceutical compositions of the invention mayalso be used in the treatment of cell proliferative diseases anddiseases involving immune deficiency, such as HIV, and of autoimmunediseases. Further, the pharmaceutical compositions of the invention maybe used for modulating the immune response during chemotherapy orradiation treatments, for example for reducing chemotherapy-associatedvomiting.

While reducing the present invention to practice, it was surprisinglyobserved that the peptides of the invention exert a synergistic effecton human hematopoietic stem cell proliferation and differentiation withaddition of other hematopoietic growth factors. Of notable significancewas the potentiation of erythropoietin-mediated stimulation of erythroidcolony formation, the potentiation of G-CSF-mediated stimulation ofgranulocyte macrophage colony formation (CFU-GM) in bone marrow cells,and the dose-dependent enhancement of thrombopoietin (TPO) induction ofmegakaryocyte proliferation by peptides of the present invention. G-CSFis currently used for mobilization of bone marrow hematopoieticprogenitor cells in donors, as a component of a wide variety of leukemiaand cancer treatments (see, for example, U.S. Pat. Nos. 6,624,154 toBenoit et al. and 6,214,863 to Bissery et al) and as a component of cellgrowth media for stem and progenitor cell manipulation (see, forexample, U.S. Pat. No. 6,548,299 to Pykett et al). Recombinant human(rh) G-CSF, marketed as Neupogen (Filgrastim, Amgen Inc., USA) has beenapproved for medical use for indications relating to neutropenia andgranulocytopenia, such as AIDS leukopenia and febrile neutropenia,respiratory and other infection (Kolls et al., Resp. Res. 2000; 2:9-11)and in chemotherapy protocols for non-myeloid malignancies. Recombinanthuman (rh) EPO is currently an approved therapy for indications such asrenal anemia, anemia of prematurity, cancer- and AIDS-associated anemia,and for pre-elective surgical treatment (Sowade, B et al. Int J Mol Med1998; 1:305).

Thus, in one preferred embodiment, a blood disease or condition such asthrombocytopenia, pancytopenia, granulocytopenia, an erythropoietintreatable condition, a thrombopoietin treatable condition, or a G-CSFtreatable condition is treated by administering to a subject in needthereof a therapeutically effective amount of a peptide derived from anN terminus portion of αS1 casein.

Further according to the present invention there is provided a method ofaugmenting the effect of erythropoietin, thrombopoietin, or G-CSF, themethod is effected by administering to a subject in need thereof atherapeutically effective amount of a peptide derived from an N terminusportion of αS1 casein. In one preferred embodiment, the method furthercomprises administering a blood cell stimulating factor such aserythropoietin, thrombopoietin, and G-CSF.

Thrombopoietin is an early acting cytokine with important multilineageeffects: TPO alone, or in combination with other early acting cytokines,can (i) promote viability and suppress apoptosis in progenitor cells;(ii) regulate hematopoietic stem cell production and function; (iii)trigger cell division of dormant multipotent cells; (iv) inducemultilineage differentiation and (v) enhance formation of multilineagecolonies containing granulocytes, erythrocytes, macrophages, andmegakaryocytes (MK, CFU-GEMM). Moreover, TPO stimulates the productionof more limited progenitors for granulocyte/monocyte, megakaryocyte anderythroid colonies, stimulates adhesion of primitive human bone marrowand megakaryocyte cells to fibronectin and fibrinogen. G-CSF is similarin action, but is specific for cells of granulocyte lineage, while EPOstimulates development of red blood cells and red blood cellprogenitors. Thus, TPO, EPO and G-CSF are important cytokines forclinical hematologists/transplanters: for the mobilization,amplification and ex vivo expansion of stem cells and committedprecursor cells for autologous and allogeneic transplantation. Inaddition, administration of TPO and G-CSF to healthy platelet donors hasbeen employed to enhance pheresis yields. However, clinical applicationof TPO, EPO and G-CSF therapy is complicated by, among otherconsiderations, relatively high costs of the recombinant human cytokinerhTPO, EPO and G-CSF and the potential antigenicity of TPO, EPO andG-CSF with repeated administration.

Combined treatment with such blood cell stimulating factor as TPO, EPOand G-CSF, and the peptide of the present invention, either together ina pharmaceutical composition comprising both, or separately, can provideinexpensive, proven non-toxic augmentation of the cytokines effects ontarget cell proliferation and function. In such a combination, thepeptide of the present invention may be applied to the treatment of, inaddition to the abovementioned conditions, disorders such asmyelodysplastic syndrome (MDS), non-myeloid malignancies, aplasticanemia and complications of liver failure. Pre-treatment of plateletdonors with the peptide of the present invention, alone or incombination with TPO and G-CSF, may even further enhance the efficiencyof pheresis yields.

Thus, according to the present invention there is provided a method ofpreventing or treating a blood disease or condition, such as athrombopoietin treatable condition, an erythropoietin treatablecondition, and a G-CSF treatable condition, the method is effected byadministering to a subject in need thereof a therapeutically effectiveamount of a peptide derived from an N terminus portion of αS1 casein.

Further according to the present invention there is provided a method ofaugmenting the effect of thrombopoietin, erythropoietin, and G-CSF, themethod is effected by administering to a subject in need thereof atherapeutically effective amount of a peptide derived from an N terminusportion of αS1 casein.

Further according to the present invention there is provided a method ofmodulating blood cell formation, the method is effected by administeringto a subject in need thereof an effective amount of a pharmaceuticalcomposition comprising effective amounts of a peptide derived from an Nterminus portion of αS1 casein alone, or in combination with blood cellstimulating factors such as thrombopoietin, erythropoietin, and G-CSF,as described hereinabove.

In one preferred embodiment, modulating blood cell formation includesinducing hematopoiesis, inducing hematopoietic stem cell proliferation,inducing hematopoietic stem cell proliferation and differentiation,inducing megakaryocytopoiesis, inducing erythropoiesis, inducingleukocytopoiesis, inducing thrombopoiesis, inducing plasma cellproliferation, inducing dendritic cell proliferation and inducingmacrophage proliferation. In a yet more preferred embodiment, thepeptide derived from an N terminus portion of αS1 casein is a syntheticpeptide, alone or in combination with peptides derived from naturalnon-αS1 casein, as described hereinabove.

Further according to the present invention there is provided apharmaceutical composition for treating a blood disease or condition,such as a thrombopoietin treatable condition, an erythropoietintreatable condition, and a G-CSF treatable condition, the pharmaceuticalcomposition comprising, as an active ingredient a peptide derived froman N terminus portion of αS1 casein and a pharmaceutically acceptablecarrier.

Further according to the present invention there is provided apharmaceutical composition for augmenting the effect of a blood cellstimulating factor, such as thrombopoietin, erythropoietin and G-CSF,the pharmaceutical composition comprising, as an active ingredient apeptide derived from an N terminus portion of αS1 casein and apharmaceutically acceptable carrier.

Further according to the present invention there is provided apharmaceutical composition for modulating blood cell formation, thepharmaceutical composition comprising, as active ingredients a peptidederived from an N terminus portion of αS1 casein alone, or incombination with blood cell stimulating factors such as thrombopoietin,erythropoietin, and G-CSF, and a pharmaceutically acceptable carrier.

In preferred embodiments, modulating blood cell formation includesinducing hematopoiesis, inducing hematopoietic stem cell proliferation,inducing hematopoietic stem cell proliferation and differentiation,inducing megakaryocytopoiesis, inducing erythropoiesis, inducingleukocytopoiesis, inducing thrombopoiesis, inducing plasma cellproliferation, inducing dendritic cell proliferation, and inducingmacrophage proliferation. Methods of monitoring the modulation of bloodcell formation, both in vivo and in vitro, are well known in the art,and are described in detail in the Examples section below.

Mobilization of stem cells from the bone marrow to the peripheralcirculation is required in a number of medical protocols. For example,in preparation for chemotherapeutic or radiation treatment ofproliferative disorders such as cancer, the patients stem cells arefirst mobilized from the bone marrow, usually via G-CSF, and collectedfor later reconstitution. Similarly, in heterologous stem cellreconstitution, the donor is treated with factors to mobilize stem cellsto the peripheral circulation prior pheresis. Methods of mobilization ofstem cells to the peripheral circulation are well known in the art (see,for example, U.S. Pat. No. 6,162,427 to Baumann et al.).

While reducing the present invention to practice, it was uncovered thatpeptides derived from an N terminus portion of αS1 casein enhanced andstimulated proliferation of hematopoietic cells in vivo and in vitro.Thus, according to the present invention there is provided a method ofenhancing peripheral stem cell mobilization, the method is effected byadministering to a subject in need thereof an effective amount of apharmaceutical composition comprising effective amounts of a peptidederived from an N terminus portion of αS1 casein alone, or incombination with blood cell stimulating factors such as thrombopoietin,erythropoietin, and G-CSF, as described hereinabove.

Further according to the present invention there is provided apharmaceutical composition for treating or preventing an indicationselected from the group consisting of hematological disease,hematological deficiencies, thrombocytopenia, pancytopenia,granulocytopenia, dendritic cell deficiencies, macrophage deficiencies,hematopoietic stem cell disorders including platelet, lymphocyte, plasmacell and neutrophil disorders, pre-leukemic conditions, leukemicconditions, myelodysplastic syndrome, non-myeloid malignancies, aplasticanemia and bone marrow insufficiency, the pharmaceutical compositioncomprising, as active ingredients, a blood cell stimulating factor suchas thrombopoietin, erythropoietin or G-CSF and a peptide derived from anN terminus portion of αS1 casein and a pharmaceutically acceptablecarrier.

Further according to the present invention there is provided apharmaceutical composition comprising a blood cell stimulating factorand a purified peptide having an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 1-23 and a pharmaceutically acceptablecarrier. In one preferred embodiment, the blood cell stimulating factoris TPO, EPO or G-CSF.

Further according to the present invention there is provided a method ofenhancing colonization of donated blood stem cells in a myeloablatedrecipient, the method is effected by treating a donor of the donatedblood stem cells with a peptide derived from an N terminus portion ofαS1 casein prior to implanting the donated blood stem cells in therecipient.

Further according to the present invention there is provided a method ofenhancing colonization of donated blood stem cells in a myeloablatedrecipient, the method is effected by treating the donated blood stemcells with a peptide derived from an N terminus portion of αS1 caseinprior to implanting the donated blood stem cells in the recipient.

Further according to the present invention there is provided a method ofenhancing colonization of blood stem cells in a myeloablated recipient,the method is effected by treating the blood stem cells with a peptidederived from an N terminus portion of αS1 casein prior to implanting theblood stem cells in the recipient. In one preferred embodiment, theblood stem cell donor, or blood stem cells, or donated blood stem cellsare further treated with a blood cell stimulating factor such asthrombopoietin, erythropoietin or G-CSF, prior to donation andimplanting the blood stem cells in the recipient. In another preferredembodiment, the peptide derived from an N terminus portion of αS1 caseinis in combination with a peptide or peptides derived from non-αS1casein.

Further according to the present invention there is provided apharmaceutical composition for enhancing colonization of donated bloodstem cells in a myeloablated recipient, the pharmaceutical compositioncomprising, as active ingredients, a peptide derived from an N terminusportion of αS1 casein and a pharmaceutically acceptable carrier.

Further according to the present invention there is provided apharmaceutical composition for enhancing colonization of blood stemcells in a myeloablated recipient, the pharmaceutical compositioncomprising, as active ingredients, a peptide derived from an N terminusportion of αS1 casein and a pharmaceutically acceptable carrier.

In one preferred embodiment, the pharmaceutical composition furthercomprises a blood cell stimulating factor such as thrombopoietin,erythropoietin or G-CSF. In another preferred embodiment, the peptidederived from an N terminus portion of αS1 casein is in combination witha peptide or peptides derived from non-αS1 casein.

The invention further relates to anti-bacterial pharmaceuticalcompositions comprising as active ingredient at least one peptide of theinvention and to the use of the peptides of the invention asanti-bacterial agents.

As detailed in the Examples section hereinbelow, peptides of theinvention, and pharmaceutical compositions comprising as an activeingredient a peptide of the invention, can be used in the treatment andprevention of blood cell disorders, cell proliferative diseases,diseases involving immune deficiency and autoimmune diseases.

Thus, according to the present invention there is provided a method ofpreventing or treating an autoimmune or infectious disease or condition,the method is effected by administering to a subject in need thereof atherapeutically effective amount of a peptide derived from an N terminusportion of αS1 casein.

In one embodiment, the autoimmune or infectious disease or condition isa viral disease, a viral infection, AIDS and infection by HIV.

Further according to the present invention there is provided a method ofpreventing or treating thrombocytopenia, the method is effected byadministering to a subject in need thereof a therapeutically effectiveamount of a peptide derived from an N terminus portion of αS1 casein.

Further according to the present invention there is provided a method ofpreventing or treating pancytopenia, the method is effected byadministering to a subject in need thereof a therapeutically effectiveamount of a peptide derived from an N terminus portion of αS1 casein.

Further according to the present invention there is provided a method ofpreventing or treating granulocytopenia, the method is effected byadministering to a subject in need thereof a therapeutically effectiveamount of a peptide derived from an N terminus portion of αS1 casein.

While reducing the present invention to practice, it was surprisinglyuncovered that administration of peptides derived from an N terminusportion of αS1 casein effectively prevented the onset of diabeticsymptoms in genetically predisposed NOD mice, and balanced bloodchemistry values in both human subjects having familialhypercholesterolemia and triglyceridemia, and in animal models. Thus,according to the present invention there is provided a method ofpreventing or treating a metabolic disease or condition, the method iseffected by administering to a subject in need thereof a therapeuticallyeffective amount of a peptide derived from an N terminus portion of αS1casein. In preferred embodiments, the metabolic disease or condition isnon-insulin dependent diabetes mellitus, insulin-dependent diabetesmellitus, glucosuria, hyperglycemia, hyperlipidemia, and/orhypercholesterolemia.

As used herein, the term “metabolic disease or condition” is defined asa deviation or deviations from homeostatic balance of metabolites in thebody, as expressed by abnormal levels of certain physiologicalparameters measurable in the body. Such physiological parameters can be,for example, hormone levels, electrolyte levels, blood glucose levels,enzyme levels, and the like.

Further according to the present invention there is provided a method ofpreventing or treating conditions associated with myeloablative doses ofchemoradiotherapy supported by autologous bone marrow or peripheralblood stem cell transplantation (ASCT) or allogeneic bone marrowtransplantation (BMT), the method is effected by administering to asubject in need thereof a therapeutically effective amount of a peptidederived from an N terminus portion of αS1 casein, alone or incombination with a blood cell stimulating factor such as thrombopoietin,erythropoietin or G-CSF.

Further according to the present invention there is provided apharmaceutical composition for preventing or treating an autoimmune orinfectious disease or condition, the pharmaceutical compositioncomprising, as an active ingredient, a peptide derived from an Nterminus portion of αS1 casein and a pharmaceutically acceptablecarrier. In preferred embodiments, the disease or condition is a viraldisease, a viral infection, AIDS, and/or infection by HIV. In furtherpreferred embodiments, the peptide of the invention is administered asan adjunct therapy, in combination with additional treatment againstviral and other infection, or to prevent onset, or reduce the severityof disease symptoms following viral infection, as in HIV and AIDStherapy.

Further according to the present invention there is provided apharmaceutical composition for preventing or treating a metabolicdisease or condition, the pharmaceutical composition comprising, as anactive ingredient, a peptide derived from an K terminus portion of αS1casein and a pharmaceutically acceptable carrier. In preferredembodiments, the metabolic disease or condition is non-insulin dependentdiabetes mellitus, insulin-dependent diabetes mellitus, glucosuria,hyperglycemia, hyperlipidemia, and/or hypercholesterolemia.

Further according to the present invention there is provided apharmaceutical composition for preventing or treating conditionsassociated with myeloablative doses of chemoradiotherapy supported byautologous bone marrow or peripheral blood stem cell transplantation(ASCT) or allogeneic bone marrow transplantation (BMT), thepharmaceutical composition comprising, as an active ingredient, apeptide derived from an N terminus portion of αS1 casein and apharmaceutically acceptable carrier.

Further according to the present invention there is disclosed the use ofa peptide derived from an N terminus portion of αS1 casein forpreventing or treating an autoimmune disease.

Further according to the present invention there is disclosed the use ofa peptide derived from an N terminus portion of αS1 casein forpreventing or treating a viral disease.

Further according to the present invention there is disclosed the use ofa peptide derived from an N terminus portion of αS1 casein forpreventing viral infection.

Further according to the present invention there is disclosed the use ofa peptide derived from an N terminus portion of αS1 casein for inducinghematopoiesis.

Further according to the present invention there is disclosed the use ofa peptide derived from an N terminus portion of αS1 casein for inducinghematopoietic stem cells proliferation.

Further according to the present invention there is disclosed the use ofa peptide derived from an N terminus portion of αS1 casein for inducinghematopoietic stem cells proliferation and differentiation.

Further according to the present invention there is disclosed the use ofa peptide derived from an N terminus portion of αS1 casein for inducingmegakaryocytopoiesis.

Further according to the present invention mere is disclosed the use ofa peptide derived from an N terminus portion of αS1 casein for inducingerythropoiesis.

Further according to the present invention there is disclosed the use ofa peptide derived from an N terminus portion of αS1 casein for inducingleukocytopoiesis.

Further according to the present invention there is disclosed the use ofa peptide derived from an N terminus portion of αS1 casein for inducingthrombocytopoiesis.

Further according to the present invention there is disclosed the use ofa peptide derived from an N terminus portion of αS1 casein for inducingplasma cell proliferation.

Further according to the present invention there is disclosed the use ofa peptide derived from an N terminus portion of αS1 casein for inducingdendritic cell proliferation.

Further according to the present invention there is disclosed the use ofa peptide derived from an N terminus portion of αS1 casein for inducingmacrophage proliferation.

Further according to the present invention there is disclosed the use ofa peptide derived from an N terminus portion of αS1 casein forpreventing or treating thrombocytopenia.

Further according to the present invention there is disclosed the use ofa peptide derived from an N terminus portion of αS1 casein forpreventing or treating pancytopenia.

Further according to the present invention there is disclosed the use ofa peptide derived from an N terminus portion of αS1 casein forpreventing or treating granulocytopenia.

Further according to the present invention there is disclosed the use ofa peptide derived from an N terminus portion of αS1 casein forpreventing or treating hyperlipidemia.

Further according to the present invention there is disclosed the use ofa peptide derived from an N terminus portion of αS1 casein forpreventing or treating cholesteremia.

Further according to the present invention there is disclosed the use ofa peptide derived from an N terminus portion of αS1 casein forpreventing or treating glucosuria.

Further according to the present invention there is disclosed the use ofa peptide derived from an N terminus portion of αS1 casein forpreventing or treating diabetes.

Further according to the present invention there is disclosed the use ofa peptide derived from an N terminus portion of αS1 casein forpreventing or treating AIDS.

Further according to the present invention there is disclosed the use ofa peptide derived from an N terminus portion of αS1 casein forpreventing or treating infection by HIV.

Further according to the present invention there is disclosed the use ofa peptide derived from an N terminus portion of αS1 casein forpreventing or treating conditions associated with myeloablative doses ofchemoradiotherapy supported by autologous bone marrow or peripheralblood stem cell transplantation (ASCT) or allogeneic bone marrowtransplantation (BMT).

Further according to the present invention there is disclosed the use ofa peptide derived from an N terminus portion of αS1 casein for treatinga thrombopoietin treatable condition.

Further according to the present invention there is disclosed the use ofa peptide derived from an N terminus portion of αS1 casein foraugmenting the effect of thrombopoietin.

Further according to the present invention there is disclosed the use ofa peptide derived from an N terminus portion of αS1 casein for enhancingperipheral stem cell mobilization.

Further according to the present invention there is disclosed the use ofa peptide derived from an N terminus portion of αS1 casein for enhancingcolonization of donated blood stem cells in a myeloablated recipient.

Further according to the present invention there is disclosed the use ofa peptide derived from an N terminus portion of αS1 casein for enhancingcolonization of blood stem cells in a myeloablated recipient.

Further according to the present invention there is disclosed the use ofa pharmaceutical composition comprising, as an active ingredient, apeptide derived from an N terminus portion of αS1 casein, and apharmaceutically acceptable carrier for preventing or treating anautoimmune disease.

Further according to the present invention there is disclosed the use ofa pharmaceutical composition comprising, as an active ingredient, apeptide derived from an N terminus portion of αS1 casein, and apharmaceutically acceptable carrier for preventing or treating a viraldisease.

Further according to the present invention there is disclosed the use ofa pharmaceutical composition comprising, as an active ingredient, apeptide derived from an N terminus portion of αS1 casein, and apharmaceutically acceptable carrier for preventing or treating a viralinfection.

Further according to the present invention there is disclosed the use ofa pharmaceutical composition comprising, as an active ingredient, apeptide derived from an N terminus portion of αS1 casein, and apharmaceutically acceptable carrier for inducing hematopoiesis.

Further according to the present invention there is disclosed the use ofa pharmaceutical composition comprising, as an active ingredient, apeptide derived from an N terminus portion of αS1 casein, and apharmaceutically acceptable carrier for inducing hematopoietic stem cellproliferation.

Further according to the present invention there is disclosed the use ofa pharmaceutical composition comprising, as an active ingredient, apeptide derived from an N terminus portion of αS1 casein, and apharmaceutically acceptable carrier for inducing hematopoietic stemcells proliferation and differentiation.

Further according to the present invention there is disclosed the use ofa pharmaceutical composition comprising, as an active ingredient, apeptide derived from an N terminus portion of αS1 casein, and apharmaceutically acceptable carrier for inducing megakaryocytopoiesis.

Further according to the present invention there is disclosed the use ofa pharmaceutical composition comprising, as an active ingredient, apeptide derived from an N terminus portion of αS1 casein, and apharmaceutically acceptable carrier for inducing erythropoiesis.

Further according to the present invention there is disclosed the use ofa pharmaceutical composition comprising, as an active ingredient, apeptide derived from an N terminus portion of αS1 casein, and apharmaceutically acceptable carrier for inducing leukocytopoiesis.

Further according to the present invention there is disclosed the use ofa pharmaceutical composition comprising, as an active ingredient, apeptide derived from an N terminus portion of αS1 casein, and apharmaceutically acceptable carrier for inducing thrombocytopoiesis.

Further according to the present invention there is disclosed the use ofa pharmaceutical composition comprising, as an active ingredient, apeptide derived from an N terminus portion of αS1 casein, and apharmaceutically acceptable carrier for inducing plasma cellproliferation.

Further according to the present invention there is disclosed the use ofa pharmaceutical composition comprising, as an active ingredient, apeptide derived from an N terminus portion of αS1 casein, and apharmaceutically acceptable carrier for inducing dendritic cellproliferation.

Further according to the present invention there is disclosed the use ofa pharmaceutical composition comprising, as an active ingredient, apeptide derived from an N terminus portion of αS1 casein, and apharmaceutically acceptable carrier for inducing macrophageproliferation.

Further according to the present invention there is disclosed the use ofa pharmaceutical composition comprising, as an active ingredient, apeptide derived from an N terminus portion of αS1 casein, and apharmaceutically acceptable carrier for preventing or treatingthrombocytopenia.

Further according to the present invention there is disclosed the use ofa pharmaceutical composition comprising, as an active ingredient, apeptide derived from an N terminus portion of αS1 casein, and apharmaceutically acceptable carrier for preventing or treatingpancytopenia.

Further according to the present invention there is disclosed the use ofa pharmaceutical composition comprising, as an active ingredient, apeptide derived from an N terminus portion of αS1 casein, and apharmaceutically acceptable carrier for preventing or treatinggranulocytopenia.

Further according to the present invention there is disclosed the use ofa pharmaceutical composition comprising, as an active ingredient, apeptide derived from an N terminus portion of αS1 casein, and apharmaceutically acceptable carrier for preventing or treatinghyperlipidemia.

Further according to the present invention there is disclosed the use ofa pharmaceutical composition comprising, as an active ingredient, apeptide derived from an N terminus portion of αS1 casein, and apharmaceutically acceptable carrier for preventing or treatingcholesteremia.

Further according to the present invention there is disclosed die use ofa pharmaceutical composition comprising, as an active ingredient, apeptide derived from an N terminus portion of αS1 casein, and apharmaceutically acceptable carrier for preventing or treatingglucosuria.

Further according to the present invention there is disclosed the use ofa pharmaceutical composition comprising, as an active ingredient, apeptide derived from an N terminus portion of αS1 casein, and apharmaceutically acceptable carrier for preventing or treating diabetes.

Further according to the present invention there is disclosed the use ofa pharmaceutical composition comprising, as an active ingredient, apeptide derived from an N terminus portion of αS1 casein, and apharmaceutically acceptable carrier for preventing or treating AIDS.

Further according to the present invention there is disclosed the use ofa pharmaceutical composition comprising, as an active ingredient, apeptide derived from an N terminus portion of αS1 casein, and apharmaceutically acceptable carrier for preventing or treating infectionby HIV.

Further according to the present invention there is disclosed the use ofa pharmaceutical composition comprising, as an active ingredient, apeptide derived from an N terminus portion of αS1 casein, and apharmaceutically acceptable carrier for preventing or treatingconditions associated with myeloablative doses of chemoradiotherapysupported by autologous bone marrow or peripheral blood stem celltransplantation (ASCT) or allogeneic bone marrow transplantation (BMT).

Further according to the present invention there is provided a purifiedpeptide having an amino acid sequence selected from the group consistingof SEQ ID NOs:1-25.

Further according to the present invention there is provided apharmaceutical composition comprising a purified peptide having an aminoacid sequence selected from the group consisting of SEQ ID NOs:1-25 anda pharmaceutically acceptable carrier.

Further according to the present invention there is provided apharmaceutical composition comprising a blood cell stimulating factor,e.g. selected from the group consisting of thrombopoietin,erythropoietin and G-CSF, and a peptide having an amino acid sequenceselected from the group consisting of SEQ ID NOs: 1-25 and apharmaceutically acceptable carrier.

The invention further relates to methods of treatment comprising theadministration of, and pharmaceutical compositions comprising,combinations of peptides derived from αS-1 casein. While reducing thepresent invention to practice, it was uncovered that combinations ofpeptides derived from αS1 casein were effective in enhancing leukocyteproliferation following bone marrow reconstitution in mice (see FIG.23). In one embodiment, the combination of peptides comprises a mixtureof peptides, not linked by covalent bond therein-between.

The invention further relates to anti-viral pharmaceutical compositionscomprising as active ingredient at least one peptide of the inventionand to the use of the peptides of the invention as anti-viral agents.While reducing the present invention to practice, it was uncovered thatpeptides derived from natural and synthetic N terminus portion of αS1casein have efficient immuno-modulatory activity that is completely freeof any demonstrable side effects.

As described in detail in the Examples section hereinbelow, peptidesderived from natural casein are capable of stimulating proliferation ofvarious types of blood stem cells and can effectively enhancereconstitution of white blood cells and platelets even in patients whoare completely resistant to platelet transfusion. Peptides derived fromnatural casein are effective in patients who are completely resistant toother modalities known to potentially enhance platelet reconstitution(including rhIL-3 and rhIL-6). Peptides derived from an N terminusportion of αS1 casein are an efficient immunomodulator capable ofenhancing hemopoietic processes of different blood stem cells with apowerful effect on White Blood Cells (WBC), platelet reconstitution andstimulation of NK activity.

Thus, according to a further aspect of the present invention there isprovided a method of treating or preventing a condition associated witha SARS infective agent, the method comprising administering to a subjectin need thereof a therapeutically effective amount of a peptide derivedfrom an N terminus portion of αS1 casein.

Further according to the present invention there is provided apharmaceutical composition for preventing or treating a conditionassociated with a SARS infective agent, the pharmaceutical compositioncomprising, as an active ingredient, a peptide derived from an Nterminus portion of αS1 casein and a pharmaceutically acceptablecarrier. In a preferred embodiment the SARS infective agent is acoronavirus. In a most preferred embodiment the coronavirus is SARS-CoV.

It will be appreciated by one of ordinary skill in the art, that theefficacy of compositions of peptides derived from natural casein forprevention and/or treatment of conditions associated with SARS infectiveagent can be evaluated both in vitro and in clinical trials. Recently,Rota et al. (Sciencexpress, 1 May 2003, see www.sciencexpress.org)reported the characterization of the SARS-CoV virus, and successfulin-vitro growth and isolation of SARS-CoV in Vero cells. Thus, forexample, as described hereinbelow for HIV-1, Vero cells can be exposedto compositions of peptides derived from natural casein both prior toand following exposure to a SARS infective agent, and the levels ofinfection can be determined, for example, via measurement of viralspecific transcripts, protein products or virion production usingmethods well known in the art.

As detailed hereinabove, the αS2, β, and κ-fractions of casein have beenshown to contain peptides having advantageous biological properties. Itwill be appreciated that combinations of peptides derived from anN-terminal portion of natural αS1-casein, and other non-αS1 caseinderived peptides (such as αS2-, β- and κ-casein) can have a synergisticeffect on the modulation and enhancement of hematopoietic,immunological, EPO-, TPO-, G-CSF-mediated, anti-viral and otherprocesses for which peptides derived from natural αS1-casein have beenshown herein to be effective. Thus, further according to the presentinvention there is provided a pharmaceutical composition comprisingpeptides derived from the N-terminus portion of αS1-casein incombination with peptides derived from non-αS1-casein, wherein saidcombination is a mixture of peptides.

While reducing the present invention to practice, a low-temperaturemethod for processing casein hydrolysate at low temperatures wasconceived. This novel method for the inactivation and removal of theprotease following digestion of the casein, is superior in rapidity andease, and without the undesirable disadvantages of traditional methodsusing heat inactivation. By replacing the high (>75° C.) heatinactivation step with cooling and alkalinization, effective andabsolute inactivation of the proteases, with no danger to the peptides,was achieved.

Thus, according to a further aspect of the present invention there isprovided a method of low-temperature processing of casein proteolytichydrolysate, the method is effected by obtaining a casein proteolytichydrolysate comprising proteolytic enzymes, cooling the caseinproteolytic hydrolysate so as to inactivate the proteolytic enzymes,adjusting the pH of the casein protein hydrolysate to an acid pH,filtering the acidic casein protein hydrolysate and collecting thefiltrate. Methods for batch cooling of the casein hydrolysate, followingproteolytic digestion, are well known in the art (see, for example,industrial fermentor and bioreactor temperature control systems fromBioGenTek, New Delhi, India) and in the dairy products industry(suitable heat exchange systems for large and small volume applicationsare widely available commercially).

The filtrate is then further acidified so as to precipitate the proteinsderived from natural casein, separated and collected, and then the pH ofthe precipitate is adjusted to an alkaline pH with a base such as NaOH,so as to irreversibly inactivate the proteolytic enzymes. Followinginactivation of die proteolytic enzymes, the pH of the precipitate isreadjusted with acid, such as HCl, to pH 7-9, thereby processing thecasein protein hydrolysate at low temperature. In a preferredembodiment, the casein hydrolysate is cooled to about 10° C., mostpreferably to 8-10° C. Temperature is maintained at 10° C. by additionof cold TCA, and centrifugation at a temperature less than 10° C.

In a further embodiment, the pH is adjusted to acid pH by addition ofacid to 2% (w/v) acid, and further acidifying the filtrate is effectedby additional addition of acid to about 10% (w/v) acid. In a preferredembodiment, the alkaline pH of the precipitate is adjusted with a baseto at least pH 9, preferably pH 10, most preferably pH 13. In preferredembodiment, alkaline pH is maintained for greater than IS minutes, morepreferred for greater than 30 minutes, and in a most preferredembodiment greater than 1 hour. Monitoring of the residual proteolyticactivity following cooling and alkaline treatment, can be used todetermine the optimal range of alkaline treatment.

As used herein, the term “about” is defined as the range comprising from20% greater than to 20% lees than the indicated value. Thus, the phrase“about 10° C.”, as used herein, includes the range of temperatures from8° C. to 12° C. Similarly, the phrase “about 10% (w/v) acid” includesthe range of acid content from 8% w/v to 12% w/v. The present inventionsuccessfully addresses the shortcomings of the presently knownconfigurations by providing peptides for the treatment of human disease,which peptides are derived from the N terminus portion of αS1 casein andposses no detectable toxicity and high therapeutic efficacy.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Materials and Experimental Methods

Preparation of peptides derived from natural casein: The casein fractionof cow's milk was isolated as described by Hipp et al. (1952), ibid., orprovided as commercial casein, and subjected to exhaustive proteolyticdigestion with chymosin (also known as rennin) (20 ng per ml) at 30° C.Upon completion of the reaction, the solution was heated to inactivatethe enzyme, and the digest was precipitated as paracaseinate byacidification with an organic acid, acetic or trichloracetic acid.Paracaseinate was separated by centrifugation, and the supernatantfraction, containing the peptide fragments of interest, wasre-precipitated as caseicidin by higher acid concentrations. Theresulting caseicidin, following re-suspension, dialysis andneutralization was lyophilized. The resulting powdered preparation wasassayed for biological activity as described below, and separated byHPLC for peptide analysis.

Alternatively, the caseicidin can be prepared by cooling and alkalinetreatment. Following digestion of the casein, the reaction mixture wascooled immediately to below 10° C. and cold TCA (Tri-chloro acetic acid)was added to obtain a 2% TCA solution. The solution was separated bycentrifugation at 1370×g, at a temperature less than 10° C.

The supernatant was removed and filtered. Additional cold TCA was addedto obtain a 10%-12.5% TCA solution. The solution was centrifuged at1370×g, at a temperature below 10° C. The precipitate was removed anddissolved in H₂O and made alkaline by a strong base, such as, forexample, NaOH, to increase the pH of the hydrolysate to pH 9-13. Thesolution was maintained at basic pH between 15 min to 1 hour.Subsequently, the solution was acidified to pH 7-9 by addition of anacid such as HCl. The resultant mixture of peptides was furtherfractionated and purified by gel filtration on a dextran column (such asSephadex), as described herein, or by diafiltration on a series of rigidmembranes, for example, using a first diafiltration apparatus with a 10kDa cutoff, and a second diafiltration apparatus with a 3 kDa cutoff(Millipore, Billerica, Mass., USA).

HPLC analysis of peptides derived from natural casein: Peptides derivedfrom natural casein as described above were analyzed by HPLC in twostages. Initially, the lyophilized casein digests were separated using aC 18 reversed phase with a 0.1% water trifluoroacetic acid(w/w)-acetonitrile gradient Detection was according to UV absorption at214 nm. Following this the samples were analyzed by HPLC-MassSpectroscopy (MS) equipped with an electrospray source. Masscalculations represent the mass of the ionized peptide samples, asderived from the retention times. Following separation, the amino acidcomposition of the peptides was determined with a gas-phasemicrosequencer (Applied Biosystems 470A).

Analysis of some preparations of peptides derived from natural caseinproduced the following results: Eight peptide peaks were typicallyobserved of which 3 were major peaks having Rt values of 17.79, 19.7,23.02 and 5 were minor peaks having Rt values of 12.68, 14.96, 16.50,21.9 and 25.1, which Rt values represent molecular mass of 2764, 6788,1880, 2616, 3217, 2333, 6708 and 6676 Da, respectively. At Rt of 17.79(corresponding to 2,764 Da) a major peak of a peptide of 23 amino acidsrepresenting amino acids 1-23 of αS1 casein, having the sequenceRPKHPIKHQGLPQEVLNENLLRF (SEQ ID NO:22, see McSweeny et al., 1993, ibid.,for the complete sequence of αS1 casein). Other peptides were frompositions 208-224 of β casein, positions 16-37 of αS1 casein andpositions 197-222 of αS2-like casein precursor. Other peptides were alsopresent. Some preparations of peptides derived from natural casein werefurther analysed with HPLC-MS (C-18 resin) and sequenced using MS/MS andEdman degradation. The column used was Vydac C-18, and the elution wascarried out with a gradient starting with 2% CH₃CN, 0.1% TFA andcontinues by increasing modifier (2% H₂O, 0.1% TFA in CH₃CN) up to 80%at 80 min. Mass Spectrometry was carried out with Qtof2 (Micromass,England), using a nanospray attachment Edman degradation was carried outusing a Perkin Elmer (Applied Biosystems Division) 492 (procise)Microsequencer system. Further HPLC-MS was also carried out using a C-12resin. Analysis of peptides derived from natural casein revealed threemajor components:

i) a peptide representing an N-terminal portion of αS1 casein, aminoacid coordinates 1-23 of the processed peptide (SEQ ID No: 22).Molecular mass is 2764 daltons.

ii) a peptide representing amino acid coordinates 193-209 of (3 casein(SEQ ID No. 26). Molecular mass is 1880 daltons.

iii) a peptide representing amino acid coordinates 106-169 of κ casein(SEQ ID No. 27). Molecular mass is 6708 daltons. The κ casein was foundin two forms: a phosphorylated form, and an un-phosphorylated form. Themolecular mass of the phosphorylated peptide is 6789 daltons. Furtherthere was identified a known variant of κ casein, whose molecular massis 6676 Da (non-phosphorylated). Four minor components were identified:

i) a peptide representing an N-terminal portion of αS1 casein, aminoacid coordinates 1-22 of the processed peptide (SEQ ID No: 23).Molecular mass is 2616 daltons.

ii) a peptide representing amino acid coordinates 165-199 of αS1 casein(SEQ ID No. 28). Molecular mass is 3918 daltons.

iii) a peptide representing amino acid coordinates 182-207 of αS2 casein(SEQ ED No. 29). Molecular mass is 3217 daltons.

iv) a peptide representing amino acid coordinates 189-207 of αS2 casein(SEQ ID No. 30). Molecular mass is 2333 daltons.

Minor peptides representing portions of the N-terminal of β casein, andother portions of bovine casein were also detected.

Gel Filtration of Peptides Derived from Natural Casein:

Peptides derived from natural casein, prepared as described hereinabove,were separated according to molecular mass by gel filtration usingSuperdex75 Gel filtration column by Pharmacia. The elution buffer usedfor the preparative separation was NH₄HCO₃, pH=8. The following purifiedfractions were obtained: a peptide representing amino acid positions1-23 of the N-terminus of αS1 casein (SEQ ID No. 22), and a secondfraction comprising a peptide representing amino acid positions 106-169of κ-casein (SEQ ID No.26). Without wishing to be limited by a singlehypothesis, one explanation for the apparent discrepancy between theanalyses of the peptides derived from natural casein by the HPLC, HPLCMS and gel filtration methods is the tendency of gel filtration toretard specific components of a mixture of peptides.

Synthetic peptides derived from casein: Peptides of increasing lengthscorresponding to the N-terminal 2-26 amino acids of αS1 casein weresynthesized by NoVetide Ltd., Haifa, Israel, with purity of >95% (HPLC).Quality Control included: HPLC, Mass Spectrometry (EI), Amino acidanalysis and Peptide Content Table 3 below provides the sequence ofthese peptides:

TABLE 3 No. of Identi- Sequence amino SEQ ID fication (N terminus - Cterminus) acids NO: 74 RP 2 1 1P RPK 3 2 2P RPKH 4 3 3P RPKHP 5 4 4PRPKHPI 6 5 5P RPKHPIK 7 6 Y RPKHPIKH 8 7 X RPKHPIKHQ 9 8 1a RPKMPIKHQO10 9 2a RPKHPIKHQGL 11 10 3a RPKHPIKHQGLP 12 11 A RPKHPIKHQGLPQ 13 12 BRPKHPIKHQGLPQE 14 13 C RPKHPIKHQGLPQEV 15 14 D RPKHPIKHQGLPQEVL 16 15 ERPKHPIKHQGLPQEVLN 17 16 F RPKHPIKHQGLPQEVLNE 18 17 C RPKHPIKHQGLPQEVLNEN19 18 H RPKHPIKHQGLPQEVLNENL 20 19 I RPKHPIKHQGLPQEVLNENLL 21 20 JRPKHPIKHQGLPQEVLNENLLR 22 21 K RPKHPIKHQGLPQEVLNENLLRF 23 22 LRPKHPIKHQGLPQEVLNENLLRFF 24 23 M RPKHPIKHQGLPQEVLNENLLRFFV 25 24 NRPKHPIKHQGLPQEVLNENLLRFFVA 26 25

Juvenile (Type I, IDDM) Diabetes in Non-Obese Diabetic (NOD) Mice:

Peptides derived from natural casein: NOD mice are a commonly used modelfor research of autoimmune disease and human Juvenile Diabetes. Six weekold female NOD mice received either one or two injections per week of100 μg of peptides derived from natural casein, for a total of 5 or 10treatments. Control mice received no treatment. The severity of diseasewas determined according to glucosuria, which was measured using Combitest sticks [Gross, D. J. et al. (1994), Diabetology, 37:1195]. Resultswere expressed as the percent of glucosuria-free mice in each sampleover a 365-day period.

Synthetic peptides derived from casein: In another experiment, 6 weekold female NOD mice received one injection per week of 100 μg ofSynthetic peptides derived from casein for a total of 5 treatments.Control mice received no treatment. Results were expressed as the numberof healthy mice in the various treated groups.

Intraperitoneal Glucose Tolerance Test (IPGTT): The glucose tolerancetest is the definitive method for investigating glucose metabolism anddiabetic tendencies in mammals. Twenty five (25) weeks after receivingSynthetic peptides derived from casein, response to a glucose load wasassessed with an intraperitoneal glucose tolerance test. Glucoseinjection consisted of 1 g/kg body weight. Glycemic values weredetermined from blood drawn prior to test (0 minutes) and 60 minutesafter loading. Plasma glucose levels were determined with a GlucoseAnalyzer 2 (Beckman Instruments, Fullerton, Calif.) and expressed asmmol/L. Normal values do not exceed 140 mmol/L.

Stimulation of Proliferation of Natural Killer (NK) Cells:

From human Peripheral Blood Stem Cells (PBSC): PBSC of G-CSF treatedsubjects were separated on a FICOLL gradient, washed twice withRPMI-1640 medium containing 10% FCS and glutamine and seeded into 1.5 mlwells with or without peptides derived from natural casein or syntheticpeptides derived from casein, as indicated, (0-500 μg per ml). Followingtwo days incubation the cells were assayed for Natural Killer activityby measuring radioactivity released from ³⁵S-labeled K562 target cells(NEG-709A, 185.00 MBq, 2.00 mCi EASYTAGth Methionine, L-[³⁵S] 43.48 TBqper mmol, 1175.0 Ci per mmol, 0.488 ml, Boston USA). Two concentrationsof effector cells (2.5×10⁵ and 5×10⁵ cells per well) were incubated with5×10³ target cells per well (effector:target cell ratios of 50:1 and100:1, respectively) in U-bottomed 96 well tissue culture plates. Thecells were incubated for 5 hours at 37° C. in 5% CO₂, 95% air andprecipitated by 5 minutes centrifugation at 1000 rpm. ³⁵S release wasmeasured in 50 μl samples of the supernatant liquid.

From murine Bone Marrow (BM) cells: Bone marrow was collected from 4untreated BALB/c and C57B1/6 mice. Bone marrow was harvested from thelong bones of front and hind limbs of the mice by injection of mediumusing a 25 Gauge needle. Aspirated cells were washed with RPMI 1640,counted in a haemocytometer and vital-stained (20 μl of cells in 380 μlacetic acid/trypan blue), then seeded in culture bottles at 2-5×10⁶cells per ml in RPMI-1640 containing 10% Fetal Calf Serum, antibioticsand glutamine with or without 100 μg per ml peptides derived fromnatural casein. The cell cultures were incubated in 5% CO₂, 95% air for12-15 days at 37° C., harvested by 10 minutes centrifugation at 1500rpm, counted, and seeded in U-bottom wells with ⁵¹Cr (Chromium-51, 740MBq, 2.00 mCi activity) or ³⁵S (NEG-709A, 185.00 MBq, 2.00 mCi EASYTAGthMethionine, L-[³⁵S] 43.48 TBq per mmol, 1175.0 Ci per mmol, 0.488 ml,Boston USA) labeled murine lymphoma (YAC) cells at either 25:1 or 50:1effector:target cell ratio. NK activity is expressed as the percentradioactivity in the cell-free supernatants.

Proliferation of human cells in culture: Peripheral blood (PB) wascollected from healthy or affected patients. Affected patients receivedno treatment other than G-CSF supplementation prior to plasmapheresis.Bone marrow (BM) cells were collected from consenting healthy patientsor affected patients in remission following chemotherapy by aspiration.Umbilical cord blood was collected during normal births. Human cells ofthe various origins were separated on a FICOLL gradient, washed twicewith RPMI-1640 medium, and seeded into 0.2 ml flat bottom tissue culturewells at the indicated concentrations with or without peptides derivedfrom natural casein or with or without synthetic peptides derived fromcasein, as indicated. All treatments, including controls, were repeatedin triplicate. Cell proliferation was measured by ³HT incorporation:radioactive thymidine was added [thymidine (methyl-[³H]) Specificactivity 5 Ci per ml 37 MBq per ml, ICN Corp.] following incubation forthe indicated number of days. Cells were then incubated 16-20 hours withthe label, harvested and washed with medium. Incorporated radioactivitywas measured in a β scintillation counter.

Proliferation of K562 leukemia and colon cancer cell lines: Colon andK532 are established lines of cancer cells grown in culture. Both celllines were grown in culture bottles in 5% CO₂, 95% air at 37° C.,harvested and washed with medium before seeding in tissue culture wellsat 4×10⁵ cells (K562) or 3×10³ cells (Colon) per well. Peptides derivedfrom natural casein were added to the wells, at the indicatedconcentrations, and after 9 (K562) or 3 (Colon) days of incubationlabeled thymidine was added as described above. Harvesting andmeasurement of radioactive uptake was as described above.

Fluorescent Antibody Detection of NK and T Cell Proliferation in HumanPeripheral Blood Stem Cells (PBSC):

Peripheral Blood Stem cells (PBSC) from human subjects receiving G-CSFtreatment were collected by plasmapheresis, separated on a FICOLLgradient, washed twice with RPMI-1640 medium containing 10% Fetal CalfSerum and incubated in culture bottles at 37° C. in 5% CO₂, 95% air withor without peptides derived from natural casein at the indicatedconcentrations. Following 10, 14 or 28 days incubation with peptidesderived from natural casein, the presence of T cells (CD₃ surfaceantigen) and NK cells (CD₅₆ surface antigen) was detected by directimmunofluorescence using anti-CD₃ fluorescent antibody (CD₃/FITC cloneUHCT₁), anti-CD₅₆ fluorescent antibody (CD₅₆/RPE clone MOC-1) (DAKO A/S,Denmark) and mouse IgG1/RPE and IgG1/FITC antibodies as a control.Detection of fluorescently tagged cells was performed using fluorescenceactivated cell sorting (FACS).

Stimulation of Hematopoiesis from Bone Marrow (BM) Cells in Culture:

Proliferation of megakaryocytes in multipotential colonies (CFU-GEMM)from murine Bone Marrow cells: Primary bone marrow cells (1×10⁵ per ml)from 8-12 week-old C3H/HeJ mice were grown in serum-free methylcellulose-IMDM medium for 8-9 days at 5% CO₂, 95% air, at 37° C. Themedium, appropriate for the growth of multipotential colonies(CFU-GEMM), contained 1% BSA (Sigma), 10⁻⁴ M thioglycerol (Sigma),2.8×10⁻⁴ M human transferrin (TF, Biological industries, Israel), 10%WEHI-CM as a source of IL-3 and 2 units per ml erythropoietin (rhEPO, R& D Systems, Minneapolis). Colonies were scored after 8-9 days using anOlympus dark field microscope. They were picked with a micropipette,cytocentrifuged and stained with May-Grunwald-Giemsa for differentialcounts. At least 700 cells were counted for each preparation.

Proliferation of Dendritic cells in CFU-GEMM: Multipotent (CFU-GEMM)colonies grown from primary bone marrow cells as described for the assayof megakaryocyte proliferation above were collected, stained and countedfor dendritic cells. At least 700 cells were counted for eachpreparation.

Proliferation of Plasma cells in CFU-GEMM: Multipotent (CFU-GEMM)colonies grown from primary bone marrow cells as described for the assayof megakaryocyte proliferation above were collected, stained and countedfor plasma cells. At least 700 cells were counted for each preparation.

Proliferation of Macrophage cells in CFU-GEMM: Multipotent (CFU-GEMM)colonies grown from primary bone marrow cells as described for the assayof megakaryocyte proliferation above were collected, stained and countedfor macrophage cells. At least 700 cells were counted for eachpreparation.

Proliferation of Red Blood Cells in CFU-GEMM: Multipotent (CFU-GEMM)colonies grown from primary bone marrow cells as described for the assayof megakaryocyte proliferation above were collected, stained and countedfor red blood cells. At least 700 cells were counted for eachpreparation.

Proliferation of Polymorphonuclear Cells (PMN) in CFU-GEMM: Multipotent(CFU-GEMM) colonies grown from primary bone marrow cells as describedfor the assay of megakaryocyte proliferation above were collected,stained and counted for polymorphonuclear cells. At least 700 cells werecounted for each preparation.

Proliferation of megakaryocyte- and erythroid forming cells from humanbone marrow and cord blood cells: A sample of bone marrow from anapparently healthy human being was processed by density gradientseparation using Histopaque-107 (Sigma Diagnostics) to obtain a purifiedpopulation of mononuclear cells (MNC). Colony assays were performed in aplating medium containing final concentrations of 0.92% methyl cellulose(4000 centripase powder, Sigma Diagnostic), rehydrated in Iscovesmodified Dulbecco's medium containing 36 mM sodium bicarbonate (Gibco),30% fetal bovine serum (FBS) (Hyclone), 0.292 mg/ml glutamine, 100 unitsper ml penicillin and 0.01 mg per ml streptomycin (BiologicalIndustries, Beit Haemek). Cord blood from normal births was collectedand prepared as mentioned above.

Colony assay medium containing 10⁵ MNC per ml was plated in triplicatewells within a 24 well tissue culture plate (Greiner), 0.33 ml per well.The cultures were incubated at 37° C. in 5% CO₂, 95% air and 55%relative humidity with or without peptides derived from natural caseinor synthetic peptides derived from casein, at the indicatedconcentrations. Plates were scored after 14 days for colonies containingmore than 50 cells. Megakaryocytes were identified by indirectimmunofluorescence using a highly specific rabbit antibody recognizinghuman platelet glycoproteins, and an FITC-conjugated goat anti-rabbitIgG. Added growth factors included 15 ng per ml leucomax (GM-CSF)(Sandoz Pharma), and 5% vol. per vol. human phyto-hemagglutinin-m (DifcoLab)-induced conditioned medium (CM) to induce development ofgranulocyte-macrophage colonies (CFU-GM). Erythropoietin (EPO) 2units/ml was used to induce formation of erythroid colonies(burst-forming unit-erythroid-BFU-E).

Alternatively, human bone marrow cells from consenting volunteer donorsor patients undergoing autologous bone marrow transplantation wereprecultured in medium containing 10-1000 μg per ml peptides derived fromnatural casein, grown in semi-solid agar, and scored forgranulocyte-macrophage hematopoietic colonies (GM-CFU) at 7 or 14 dayspost treatment.

Megakaryocytopoiesis was measured in normal bone marrow cells fromhealthy consenting human donors by either scoring of the number ofmegakaryocytes in samples of liquid culture (RPMI-1640 plus 10% human ABserum, glutamine and antibiotics) with or without 100 μg per ml peptidesderived from natural casein, or in a methylcellulose assay for assessingcolony formation. 2×10⁵ bone marrow cells were seeded in the presence ofa standard growth factor combination with or without peptides derivedfrom natural casein. In the methylcellulose assay megakaryocytes werecounted with an inverted microscope on days 12-14 after seeding.

Clinical trials using peptides derived from natural casein: In oneseries of trials, a single dose containing 50 mg peptides derived fromnatural casein was administered intra-muscular to human subjects in 3depots, over a period of 2 hours. Clinical parameters were monitored atthe indicated intervals. In other trials, patients at various stages oftreatment for and/or remission from cancer and metastatic diseasereceived peptides derived from natural casein once or twice, and weremonitored for changes in the cell count of peripheral blood.

Inhibition of In Vitro HIV Infection of Human Lymphocyte Cells:

Peptides: Peptides [either peptides derived from natural casein orsynthetic peptides derived from casein (2-26 amino acids in length, seetable 3)] supplied as lyophilized powder were resuspended in RPMIcomplete medium and added to cell cultures at a final concentrations of50 to 1000 μg per ml.

Cells: Several types of freshly isolated human cells (primary cells) andcell lines are known to be susceptible to in vitro HIV-1 infection,although essentially any cell displaying even low surface levels of theCD₄ molecule can be considered a potential target for HIV-1 infection.Two commonly used human cell lines which are highly sensitive for HIV-1infection were chosen, CEM and Sup-T1.

CEM is a human T4-lymphoblastoid cell line initially derived by G. E.Foley et al. [(1965), Cancer 18:522] from peripheral blood buffy coat ofa 4-year old Caucasian female with acute lymphoblastic leukemia. Thesecells were continuously maintained in suspension in medium, and havebeen used widely for analysis of infectivity, antiviral agents andneutralizing antibodies.

Sup-T1 is a human T-lymphoblastoid cell line isolated from a pleuraleffusion of an 8-year old male with Non-Hodgkin's T-cell lymphoma[Smith, S. D. et al. [(1984) Cancer Research 44:5657]. This cellexpresses high levels of surface CD₄ and is useful in studies of cellfusion, cytopathic effect and infectivity of HIV-1. Sup-T1 cells aregrown in suspension in enriched medium.

Medium: Cells were grown in RPMI-1640 complete medium enriched with 10%Fetal bovine serum, 2 mM glutamine and 2 mM penicillin-streptomycin(GIBCO).

Virus: The HIV virus strain employed was HIV-1IIIB, originallydesignated HTLV-IIIB. Concentrated culture fluids of peripheral bloodfrom several patients with AIDS or related diseases were used toestablish a permanent productive infection in H-9 cells. This subtype Bvirus has high capacity to replicate in human T-cell lines. Viral titerwas 5.38 ng per ml in stock solution.

FITC-labeled peptides: FITC F-1300 (Fluorescein isothiocyanate, isomerI, Sigma (F25o-2) St, Louis, Mich., USA) having excitation/emissionmaxima of about 494/520 nm, respectively, was employed. Theamine-reactive fluorescein derivative is probably the most commonfluorescent derivatization reagent for covalently labeling proteins.FITC-conjugated peptides derived from natural casein were prepared bycovalent binding of FITC to the amine groups of lysine.

HIV-1 P²⁴ antigen capture assay: An HIV-1 P²⁴ Antigen capture assay kitemployed was designed to quantitate the HIV-1 P²⁴ core antigen, which isproportionally related to the degree of viral production in cells. Thiskit was purchased from the AIDS Vaccine program of theSAIC-NCI-Frederick Cancer Research Institute, P.O. Box B, Frederick, Md.21702, USA and included 96 well plates coated with monoclonal antibodyto HIV-1 P²⁴, primary antibody-rabbit anti-HIV P²⁴ serum, secondaryantibody-Goat anti-rabbit-IgG (H+L) peroxidase conjugated antibody, TMBperoxidase substrate system and lysed HIV-1 P²⁴ standard. The HIV-1 P²⁴antigen capture assay was analyzed by Organon-Technica ELISA reader at450 nm with a reference at 650 nm.

HIV-1 P²⁴ antigen capture ELISA: HIV infection was measured with anindirect enzyme immunoassay which detects HIV-1 P²⁴ core antigens intissue culture media. Tissue culture supernatant was reacted withprimary rabbit anti-HIV-1 P²⁴ antigen and visualized by peroxidaseconjugated goat anti rabbit IgG. The reaction was terminated by adding4N H₂SO₄, wherein the intensity of the color developed is proportionalto the amount of HIV-1 antigen present in the tissue culturesupernatant.

Biological hazard level 3 (BL-3) laboratory: All virus productionisolation and infection, tissue culture of HIV-1 infected cells, P²⁴antigen containing supernatant harvesting and P²⁴ antigen capture ELISA,were performed in BL-3 facility and were in accordance with the biosafety practices set by the NTH and CDC (USA).

Flow cytometry: A FACSort cell sorter (Becton & Dickinson, San Jose,Calif. USA) was used to (i) determine the percentage of CD₄ positive CEMand sup-T1 cells batches before infection with HIV-1 in order to assurethe same degree of infection in each experiment; and (ii) detect T cellsthat harbor FITC conjugated peptides derived from natural casein intheir cytoplasm and nuclei.

CO₂ incubator: For viral culture production cells with HIV-1, cells andvirus pretreated with peptides derived from natural casein and cellswhich were further incubated with HIV-1, were all kept in humidified CO₂incubator for the duration of the experiment.

HIV infection of human cultured CD4 cells: For longer incubations, thecells (CEM, Sup-T1) were preincubated with several increasingconcentrations of peptides derived from natural casein (50-1000 μg perml) or synthetic peptides derived from casein (10-500 μg per ml) for 24(for synthetic and natural peptides) and 48 (only for natural peptides)hours and HIV-1IIIB (45 pg per ml final concentration) was added to eachwell thereafter. For the shorter incubations (3 hours), HIV-1IIIB waspreincubated with the peptides for 3 hours and then added to cells (5000cells/well) in tissue culture plates. Controls were IF (Infected, cellscultured with HIV-1 and without peptides), UIF (Uninfected, cellscultured without HIV-1 and without peptides) and UIF+Ch(Uninfected+peptides derived from natural casein, cells cultured in thepresence of peptides derived from natural casein {50-1000 μg per ml}) totest the effect of peptides derived from natural casein and syntheticpeptides derived from casein on cell viability and growth. Cells werecounted for viability and proliferation rate on day 7, 10 and day 14post infection (the day of P²⁴ antigen culture supernatant harvest).Cells and tissue culture supernatants (media) were harvested and lysedimmediately in 1/10 volume of 10% Triton X-100. These samples werefurther incubated at 37° C. for 1 hour and kept at −80° C. until testedfor P²⁴ antigen.

Confocal microscopy: A Zeiss LSM 410 confocal laser scanning systemattached to TW Zeiss Axiovert 135M inverted microscope, employing thelaser scanning confocal microscopy technique, was used to detectpenetration of FITC conjugated peptides into cells. T cells wereincubated with FITC conjugated peptides derived from natural casein in a5% CO₂, 95% air, 37° C. incubator, after which the cells were washed 3times with phosphate buffer saline (PBS) to remove unboundFITC-peptides. Cells were fixed with 3.8% formalin for 10 minutes,washed twice with PBS and resuspended in 50-100 μl PBS before viewingthe cells under the microscope. Selected images of cells from differenttime points of incubation (15 minutes, 30 minutes, 1 hour, 1.5 hour and3 hours) displaying various amounts of FITC-peptides derived fromnatural casein in their cytoplasms and nuclei were stored on 3.5″ Zipdrive (230 MB) and processed for pictures using Photoshop software.

[³H]-thymidine incorporation test: In order to test the effect ofpeptides derived from natural casein on T cell proliferation, severalconcentrations of peptides derived from natural casein (10 mg/ml stockin RPMI) were added to Sup-T1 cell cultures in 96 flat bottom microwellplate (5000 cells/well), as described for HIV-1 infection in Sup-T1cells. Cells were counted and their viability was determined by trypanblue dye exclusion. They were pulsed with [³H]-thymidine at each timepoint (3, 7, 10 and 14 days) for 18 hours (over night) and harvested onglass fiber filters for radioactivity reading (Incorporation of[³H]-thymidine into cellular DNA is proportional to degree of cellproliferation).

Toxicity of peptides derived from natural casein in normal, myeloablatedand transplant recipient mice and guinea pigs: Intramuscular, orintravenous injections of up to 5,000 mg peptides derived from naturalcasein per kg animal were administered in a single dose, or in threedoses to normal animals. A variety of strains were employed, includingBALB/c, C3H/HeJ and Non-Obese Diabetic (NOD) mice. The mice were eithermonitored for 10 months before sacrifice and post-mortem examination(toxicity assay) or observed for 200 days (survival rate). Guinea pigsreceived a single intramuscular injection of 20 mg peptides derived fromnatural casein per animal. Fifteen days later they were sacrificed andexamined for pathology.

Leukocyte and platelet reconstitution in bone marrow transplantrecipient mice: BALB/c mice were sub-lethally irradiated at a source toskin distance of 70 cm, dosage of 50 cGy per minute, for a total of 600cGy. The irradiated mice were reconstituted with syngeneic bone marrowas described above and injected intravenously 24 hours later with 1 mgper animal peptides derived from natural casein, synthetic peptidesderived from casein (13-26 amino acids, see Table 3 above), or humanserum albumin (controls), following a double-blinded protocol. Leukocytereconstitution was determined according to cell count in peripheralblood collected at indicated intervals from 6 to 12 days post treatment.Platelet reconstitution was determined by cell count in blood collectedfrom the retro orbital plexus, into EDTA-containing vials, at indicatedintervals from day 6 to day 15 post treatment.

In an additional series of experiments, CBA mice were lethallyirradiated (900 cGy), reconstituted with BM cells and treated withpeptides derived from natural casein or human serum albumin as describedabove. Platelet reconstitution was assayed as mentioned above.

In a third series of experiments, the mice were irradiated (800 cGy),reconstituted and injected intraperitoneally with 1.0 mg syntheticpeptides derived from casein (peptides 3a and 4P, representing the first6 and 12 amino acids of the N terminus of αS1 casein, respectively—seeTable 3 above) daily, on days 4, 5, 6 and 7 post-transplantation.Platelet reconstitution was assayed at 10 and 12 dayspost-transplantation.

In a fourth series of experiments, F1 mice were irradiated (750 cGy),reconstituted with syngeneic bone marrow, and injected intravenously 24hours later with 1 mg per mouse of synthetic peptides derived fromcasein representing amino acids 1-22 of the N terminus of αS1 casein. Inaddition, 2 (two) groups of mice were treated each with a naturalfraction of αS1 casein position 1-23, and a fraction of peptides derivedfrom natural κ-casein, representing amino acid coordinates 106-169 ofκ-casein (SEQ ID No. 26). WBC counts were conducted on days 5, 7, 10 and12 post-transplantation.

Reconstitution of Bone Marrow Transplant Recipient Mice and Enhancementof Bone Marrow Cell Proliferation in Donor Mice:

C57B1/6 mice were lethally irradiated at a source to skin distance of 70cm, dosage of 50 cGy per minute, for a total of 900 cGy. The irradiatedmice were reconstituted with syngeneic bone marrow cells from mice whichwere either treated a day prior to bone marrow collection with 1 mg peranimal peptides derived from natural casein or with saline (controls),following a double-blinded protocol. In one experiment mice survival wasmonitored for 18 days. In another experiment mice were sacrificed after8 days and spleen colonization monitored.

Synthetic Peptides Derived from Casein Significantly Reduce CholesterolLevels:

The ability of synthetic casein derived peptides to reduce cholesterollevels in 7-week old female C57B1/6j mice was assessed after feeding anatherogenic diet. The mice were divided into groups of 8. One controlgroup was fed a normal diet. A second control group was fed the modifiedThomas Hartroft diet containing cholate (#TD 88051: Teklad, Madison,Wis.) [Gerber, D. W. et al., Journal of Lipid Research. 42, 2001]. Theremaining experimental groups were all fed the modified Thomas Hartroftdiet. After one week on the diet, serum cholesterol values increasedsignificantly and the synthetic peptides derived from casein wereinjected intraperitoneally, 1 mg per mouse, followed by a secondinjection of 0.1 mg one week later.

Cholesterol blood levels were determined according to Roche CholesterolAssay based on Roeschlou & Allin enzymatic method (Roche, Inc.,Germany).

EXPERIMENTAL RESULTS

Peptides derived from natural casein: Originating from the observationthat curdled milk occasionally failed to support bacterial growth, acasein fragment possessing bacteriocidal properties was isolated frommilk proteins (U.S. Pat. No. 3,764,670 to Katzirkatchalsky, et al.).Crude peptides derived by proteolysis of natural casein were prepared byacid precipitation of the soluble fraction of the casein proteolyticdigest, dialysis and lyophilization. When tested for biological activityafter extended storage, it was noted that this crude preparation, whenlyophilized and stored at 4° C., remained active (in vitro and in vivo)for at least 24 months.

Low Temperature-Processed Peptides from natural casein: Preparation ofthe Casein Hydrolysate According to Traditional Methods, Such as thatdescribed by Hipp et al. (ibid), requires high temperature (>75° C.)inactivation of the proteolytic enzymes, a time consuming processresulting in irreversible denaturation of the large amounts ofproteolytic enzymes required for the production of peptides from naturalcasein, and potential unknown effects on the hydrolysate itself. Whilereducing the present invention to practice, it was surprisinglydiscovered that the proteolytic process producing peptides from naturalcasein can be terminated more efficiently, by a novel, simplified methodcomprising cooling, alkaline treatment, and subsequent acidification.

In a representative preparation, and to compare Low-TemperatureProcessing with the conventional heat treatment, a 1.7% casein solutionprepared as described hereinabove was subjected to proteolytic digestionwith a proteolytic enzyme (for example, chymosin (known also as renin)either as crystalline renin or commercial chymosin of non-animal source.Other proteolytic enzymes, as pepsin, can also be used).

20 ng of the enzyme was added per each ml of the 1.7% casein solution.Proteolytic digestion of the casein was completed after 14.5 hours at30° C.

At the completion of the reaction, the reaction mixture was cooledimmediately to below 10° C., made 2% with cold TCA (Tri-chloro aceticacid), and maintained below 10° C. Following removal and filtration ofthe resulting supernatant, which still contained most of the peptidesderived from natural casein, the supernatant was made 10-12.5% in coldTCA, and centrifuged at 1370×g at below 10° C.

The resulting precipitate comprising peptides derived from naturalcasein was removed and dissolved in H₂O and made strongly basic (pH9-13) with an alkaline solution. The solution was kept at this basic pHbetween 15 minutes to 1 hour, and then acidified with HCl, to a final pHof between pH 7-9.

Surprisingly, it was observed that maintaining the solution at analkaline pH (between pH 9-13) for sufficient time (from 15 minutes to 1hour), terminated enzymatic activity completely, and caused anirreversible denaturation thereof. Further purification of peptides wasperformed by gel filtration or diafiltration, as described hereinabove.

In order to identify the active peptides contained in the peptidesderived from natural casein the lyophilized preparation was fractionatedusing high performance liquid chromatography (HPLC), as describedhereinabove. All of the lyophilized samples analyzed demonstratedsimilar retention time profiles, with contents as described above.

Thus, major components of the peptides derived from natural caseinpreparation are the N-terminal fragment of αS1 casein, a peptiderepresenting a fragment of β casein, and a peptide representing afragment of κ casein. Minor components identified are a fragment of theN-terminal portion of αS1 casein, a peptide representing a further,distinct fragment of αS1 casein, a peptide representing a fragment ofαS2 casein, and a peptide representing a further, distinct fragment ofαS2 casein.

Peptides derived from natural casein are non-toxic in rodents andhumans: Extensive investigation of the short and long term effects ofhigh doses of peptides derived from natural casein on mice, rats, guineapigs and human volunteers confirmed the absence of toxicity,teratogenicity or adverse side effects of the preparation. In one seriesof tests, single doses representing 7,000 times the estimated effectivedose of peptides derived from natural casein were administered intramuscularly to mice. Standard post-mortem pathology examination of themice at 14 days post treatment revealed no toxic effects on internalorgans or other abnormalities. Similar toxicity tests in guinea pigsrevealed no abnormalities two weeks after single 20 mg infra-musculardoses of peptides derived from natural casein. In another series ofexperiments, high doses of peptides derived from natural caseinadministered to healthy mice had no effect on several hematologicalparameters measured two weeks later, including white blood cells (WBC),red blood cells (RBC), hemoglobin (HGB), electrolytes, glucose andothers. A third series of experiments tested repeated high doses of 100mg per kg body weight in mice and rats for two weeks, revealing noallergic, delayed cutaneous or anaphylactic responses and nopathological effects upon post-mortem examination. When peptides derivedfrom natural casein were tested for their effect on the long-termsurvival of irradiated, bone marrow reconstituted BALB/c and C3H/HeJmice, survival of the treated mice (18 of 27 BALB/c and C3H/HeJ; 66%)clearly exceeded the survival rates of the albumin-treated controls (4of 26 BALB/c and C3H/HeJ; 15%). Standard teratogenicity tests [fordetails see, for example, Drug Safety in Pregnancy, Folb and Dakes, p.336, Elsevier; Amsterdam, N.Y., Oxford (1990)] in mice treated withpeptides derived from natural casein revealed no effect of the peptideson any developmental parameters.

Similar to its lack of toxicity or side effects when tested in rodents,peptides derived from natural casein were safe when administered tohumans as well. Comparison of blood and urine samples from seven healthyhuman volunteers before, during and 7 days after intramuscular injectionof peptides derived from natural casein revealed no changes in any ofthe clinical parameters. No other negative effects were observed.

Thus, high dose and extended treatment of rodents with peptides derivedfrom natural casein revealed no apparent toxic, pathological,hypersensitivity, teratogenic, serological or any other negativeeffects. Moreover, peptides derived from natural casein administrationto irradiated mice, at risk for short- and long-term complications,conferred a significant survival advantage over 200-300 days. These, andthe absence of any undesirable effects in healthy human volunteersreceiving peptides derived from natural casein via injections clearlydemonstrate the peptide's safety in parenteral administration.

Reconstitution of bone marrow in transplant recipient mice: When C57B1/6mice were lethally irradiated and reconstituted with syngeneic bonemarrow from mice that were either treated a day prior to bone marrowcollection with 1 mg per animal peptides derived from natural casein ornot so treated, survival of irradiated mice that received bone marrowfrom treated mice far exceeded that of irradiated mice that receivedbone marrow from non treated mice (survival of irradiated mice matreceived bone marrow from treated mice was 15 out of 18, 10 days postirradiation; whereas survival of irradiated mice that received bonemarrow cells from saline-treated control mice was 4 out of 17, 10 dayspost irradiation). Spleens derived from irradiated mice that receivedbone marrow from treated mice included about twice to three times asmany colonies per spleen, as compared to spleens of irradiated mice thatreceived bone marrow cells from saline-treated control mice (1-5colonies as compared to 0-3 colonies).

Peptides derived from natural casein stimulate the proliferation oflymphocytes: Natural killer (NK) and cytotoxic T cells are crucial tothe immune system's ability to protect against invasion by bothinfectious pathogens and cancer cells, by both active cytotoxicity andthe secretion of immunoregulatory lymphokines. Immune compromise, suchas in AIDS or following chemotherapy, results in abnormal, weakened T orNK cell activity. When normal murine bone marrow cells from BALB/c andC57B1/6 mice were cultured in the presence of 100 μg per ml peptidesderived from natural casein, a clear increase in NK activity wasobserved in both effector:target cell ratio groups. Moreover, comparisonbetween the two groups revealed a clear dose response relationship. Atthe 25:1 effector:target cell ratio the average NK activity was elevatedfrom 13.93% to 30.77% and at the 50:1 effector:target cell ratio theaverage NK activity was elevated from 13.68% to 44.05% (FIG. 1). Similarexperiments using human Peripheral Blood Stem Cells from GranulocyteColony Stimulating Factor-treated donors demonstrated an even moresignificant, concentration-dependent stimulation of target cell lysis bypeptides derived from natural casein.

In the first set of experiments (FIG. 2 a), NK activity was measured inblood samples taken from one patient and incubated at twoeffector:target cell ratios with increasing peptides derived fromnatural casein concentration. Only 4% ³⁵S release was measured in thecontrol, untreated PBSC culture. Almost the same percent radioactivity(4%) was found at the lowest peptide concentration (5 μg per ml).However, at higher peptide concentrations, in the range of 10 μg per mlup to 100 μg per ml, a release of 10.8-14.9% ³⁵S was measured foreffector:target cell ratios of 100:1 and 8.3-14.5% ³⁵S foreffector:target cell ratios of 50:1 (FIG. 2 a).

When PBS cells from normal (patient 1) and affected (patients 2-6) humandonors were incubated with increasing concentrations of the peptidesderived from natural casein, a significant enhancement of affectedpatients' NK cell activity could be measured. Thus, while the peptidesderived from natural casein had a minimal effect on the normal patient'sNK activity (increased from 13-15% ³⁵S release, patient 1), PBS cellsfrom both breast cancer and Non-Hodgkins Lymphoma patients (patients 3and 4, for example) exhibited dramatic, dose-dependent increases in NKactivity (3.5 to 10.8% ³⁵S; 12.2 to 19.1% ³⁵S, respectively) (FIG. 2 b).

Peptides derived from natural casein stimulate the proliferation of CD56surface antigen positive (NK) cells: In another series of experimentsPeripheral Blood Stem Cells (PBSC) from 5 human donors receiving G-CSFtreatment were incubated with peptides derived from natural casein for10, 14, or 28 days, then assayed for presence of the CD₅₆ antigen. Asometimes dramatic increase in CD₅₆ antigen detection was observed inthe peptide-treated cells from all the donors but one (patient 1). Arepresentative response is depicted in FIG. 3 a: Following 10 days ofincubation with or without peptides derived from natural casein, thepresence of CD56 surface antigen-positive (NK) cells was detected bydirect immunofluorescent staining. Overall, incubation with peptidesderived from natural casein increased the mean percentage of the cellspositively stained for CD56 from 0.64% in the control group to 2.0%following treatment (FIG. 3 a).

Peptides derived from natural casein stimulate the proliferation of CD3surface antigen-positive (T) cells: The effect of peptides derived fromnatural casein on the proliferation of CD3 surface antigen-positive (T)cells in PBS cells from 5 subjects was assayed by directimmunofluorescence. In all but one patient (patient 4), 14 daysincubation with peptides derived from natural casein significantlyincreased T-cell proliferation, up to more than 5 fold in some. Takentogether, the mean percentage of the cells positively stained for CD3increased from 19.45% in the control group to 35.54% in the treatedgroup (FIG. 3 b).

Peptides derived from natural casein stimulate the proliferation of—CD56and CD3 (NK/T-cells) positive cells: In an additional experiment PBSCsfrom 7 patients were incubated with peptides derived from natural caseinfor 28 days, and the effect on proliferation of NK/T cells (CD56 and CD3surface antigen-positive) was detected by direct immunofluorescence.Incubation with peptides derived from natural casein stimulatedproliferation of T-cell greater than 5 fold in some cases (patient 6),while the mean percentage of the CD3-positive (T-) cells increased from2.08% in the control group to 6.49% in the treated group. The number ofboth CD56 and CD3 surface antigen-positive (NK/T) cells was increasedfrom 1.1% in the control to 4.3% in the treated group (FIG. 3 c). Thus,peptides derived from natural casein stimulate the proliferation of bothT-lymphocytes and Natural Killer cells from normal murine and humanblood cell progenitors. Significantly, the greatest immune-stimulatoryeffect of the peptides derived from natural casein was noted in humandonors having initially low T- and NK cell levels (FIGS. 3 a-c).

Synthetic peptides derived from casein stimulate human lymphocyteproliferation in vitro: When synthetic peptides derived from anN-terminus portion of αS1 casein representing the first 3 to 26 residuesof αS1 casein were incubated with human PBSC cells from healthy andcancer patients (see below), a significant increase in NK cell activitywas observed. Target cell lysis was greatest (from 3 to greater than 5fold that of controls) in Non-Hodgkin's Lymphoma and Breast Cancerpatient's PBSC cultures after two days incubation with as little as 10μg per ml of peptides containing the first 9 or more residues of αS1casein (FIG. 4). Under identical conditions, none of the peptides testedhad a significant effect on NK activity in PBSC cultures from healthyhuman donors. Thus, even low concentrations of peptides containing thefirst 10 residues of the N-terminal sequence of αS1 casein are capableof selectively stimulating in vitro lymphocyte proliferation in cellsfrom cancer patients.

Similar stimulation of NK cell activity was observed when PBS cells fromhuman donors with hematopoietic disease were incubated with syntheticpeptides derived from αS1 casein representing the first 3 amino acidresidues of αS1 casein. Incubation of the PBS cells with the peptidesincreased target cell lysis from 2- to greater then 8-fold that of theuntreated controls. Of the 5 patients tested, three (3) responded to 25μg/ml peptide concentration, one (1) responded to 100 μg/ml peptideconcentration and one (1) to 250 μg/ml. No significant effect on NKactivity in PBSC cultures from healthy human donors treated with thesynthetic peptide representing the first 3 amino acids of αS1 casein,was observed, confirming the selective nature of the humanlymphocyte-stimulating properties of αS1 casein-derived peptides.

Stimulation of Hematopoiesis in Human Blood Cell Progenitors:

Blood cell progenitors differentiate into a variety of blood cells:macrophages, monocytes, granulocytes, lymphocytes, erythrocytes andmegakaryocytes. Progenitor cells are abundant in bone marrow, but arealso found in peripheral blood after Granulocyte Colony StimulatingFactor treatment (PBSC cells), and fresh Cord Blood. When increasingconcentrations (50-600 μg per ml) of peptides derived from naturalcasein were added to cultures of human Bone Marrow, PBSC and Cord Blood,an increase in cell proliferation, as measured by [³H]-thymidineincorporation was noted (FIGS. 5 a-5 c). Human PBSC proliferation wasmost greatly effected by 300 μg per ml (FIG. 5 a) after 15 days inculture. An even greater effect was noted for Cord Blood cells inculture (3 to 4 fold increase in [³H]-thymidine incorporation) after 14days incubation (but not after 7 days) with peptides derived fromnatural casein (600 μg per ml, FIG. 5 c). Cultured human bone marrowcells from three out of four donors also reacted strongly (3 to 5 foldincrease in incorporation) to peptides derived from natural casein (300μg per ml) after 21 days incubation (FIG. 5 b). Thus, peptides derivedfrom natural casein stimulate proliferation of human blood cellprogenitors from bone marrow as well as other sources. Interestingly,incubation of cultured human K562 (Chronic Myeloid Leukemia) and Colon(Colon cancer) cell lines with high concentrations (up to 500 μg per ml)of peptides derived from natural casein under similar conditions had noeffect on [³H]-thymidine incorporation. Thus, peptides derived fromnatural casein exhibited a selective effect on cell proliferation,stimulating proliferation of human blood cell progenitors but not growthof cancerous cells in vitro.

Stimulation of Megakaryocytopoiesis by Peptides Derived from Casein:

Peptides derived from natural casein stimulate megakaryocyte progenitorproliferation in cultured murine bone marrow cells: Multinucleatedmegakaryocytes develop in the bone marrow from primitive stem cells,mature to giant cells and give rise to thousands of thrombocytes permegakaryocyte. Thrombocytes are crucial for clot formation andthrombocytopenia is a major concern in myeloablative conditions(following chemotherapy or radiotherapy).

Primary bone marrow cell cultures can be induced to form CFU-GM(Granulocyte and Monocyte) colonies, and CFU-GEMM (Granulocyte,Erythroid, Macrophage and Megakaryocyte) colonies, containing additionalblood cell types. Colony counts reflect expansion of specificprogenitors, cell numbers reflect proliferation rates and differentialcell counts reflect which specific cell lineages have developed[Patenkin, D. et al. (1990), Mol. Cel. Biol. 10, 6046-50]. In culturedmurine bone marrow cells incubated with erythropoietin and IL-3,addition of 25 μg per ml peptides derived from natural casein for 8 daysincreased the number of CFU-GEMM two and one half fold over controls,stimulating a three fold increase in relative cell numbers per colony inthe CFU-GEMM. In a similar series of experiments, addition of peptidesderived from natural casein to bone marrow cells incubated witherythropoietin and conditioned medium (see Materials and ExperimentalMethods) stimulated a concentration-dependent increase in the percentageof early and late megakaryocytes (15% megakaryocytes without peptides,to 50% with 500 μg per ml peptides derived from natural casein). Thus, 8days treatment with peptides derived from natural casein stimulated asignificant increase in megakaryocyte formation and development inprimary murine bone marrow cultures.

Synthetic Peptides Derived from Casein Stimulate MegakaryocyteProgenitor Proliferation in Cultured Murine Bone Marrow Cells:

Similar to the above and under similar experimental conditions,synthetic peptides derived from casein representing the first 5 to 24amino acids of αS1 casein increase the percentage of early and latemegakaryocytes from 15% without the synthetic peptide to more than 40%with 25 μg per ml of synthetic peptides (FIG. 7). Thus, 8 days treatmentwith synthetic casein derived peptides representing the first 5, 6, 11,12, 17, 18, 19, 20, 21 and 24 amino acids stimulated a significantincrease in megakaryocyte formation and development in primary murinebone marrow culture. Somewhat milder, yet appreciable, stimulation wasobserved with the other synthetic peptides derived from αS1 casein.

In a similar experimental regimen, synthetic peptides representing aminoacids 1-22 of αS1-casein (SEQ ID NOs. 21) also stimulated an increase inearly, late and total megakaryocyte formation and development in primarymurine bone marrow cultures. An increase in total megakaryocyteproliferation of 57% over controls was observed in cells supplementedwith synthetic αS1-casein (FIG. 21).

Peptides derived from natural casein stimulate Megakaryocytopoiesis incultured human bone marrow cells: When 100 μg per ml peptides derivedfrom natural casein were added under similar conditions to human bonemarrow cell cultures from healthy donors, CFU-GM colony formation wasincreased with or without additional stimulating factors (GM-CSF, CM).Peptides derived from natural casein also stimulated erythroid cellforming colonies in the presence of erythropoietin. Treatment of thehuman bone marrow cells with thrombopoietin (TPO) stimulatesmegakaryocyte (MK) colony formation. Addition of 300 μg per ml peptidesderived from natural casein to TPO-treated cells stimulates a more thantwofold increase (16 colonies per 2×10⁵ cells without peptides, 35colonies per 2×10⁵ with peptides derived from natural casein) in MKcolony proliferation.

In the presence of additional hematopoietic factors, such aserythropoietin, human IL-3, hSCF and AB serum, 14 days incubation withpeptides derived from natural casein stimulated a nearly three foldincrease in CFU-GEMM colonies from human bone marrow cells (158 colonieswith 500 μg per ml peptides derived from natural casein, 68 colonieswith the factors alone), but had a smaller (one and one half fold)effect on cultured cord blood CFU-GEMM formation. The relative cellnumber counts in the cultured human bone marrow and cord blood coloniesreflect megakaryocyte cell proliferation in response to addition of 25μg per ml peptides derived from natural casein (see Table shown in FIG.6). Thus, incubation of cultured human primary bone marrow and cordblood cells with peptides derived from natural casein stimulates thedevelopment and proliferation of both committed megakaryocyte anderythroid cell colonies. Significantly, the synergy observed between TPOand peptides derived from natural casein in stimulatingmegakaryocytopoiesis indicates a probable role for this potenthematopoietic growth factor in the mechanism of peptides derived fromcasein's stimulatory properties, and further suggests the likelihood ofsimilar augmentation of a wide range of TPO-mediated effects by peptidesderived from natural casein.

Peptides derived from natural casein and synthetic peptides derived fromnatural casein potentiate the effect of Erythropoietin (EPO) in culturedhuman bone marrow cells: The effect of natural and synthetic peptidesderived from casein on erythroid cell proliferation in cultured humanbone marrow cells was assessed under the same conditions outlinedhereinabove for megakaryocytopoiesis. When added in the presence of EPO,50-300 μg/ml peptides derived from natural casein, or 100 μg/mlSynthetic peptides derived from casein (F, Table 3, SEQ ID NO:18)stimulated a one and one-half (synthetic peptide) to four-foldproliferation of erythroid cell precursors (appearance of BFU-Ecolonies) compared to the bone marrow cells treated with EPO alone.Thus, peptides derived from natural casein and synthetic derivativesthereof act to potentiate the erythropoietic-stimulating effects of EPO,and as such can be used to augment of a wide range of clinicallyimportant EPO-mediated effects.

Synthetic peptides derived from casein stimulate Dendritic cellsproliferation in murine CFU-GEMM: The effect of Synthetic peptidesderived from αS1 casein on dendritic cell proliferation in murineprimary bone marrow cells was assessed under the same conditionsoutlined for the stimulation of megakaryocytes. Synthetic peptidesderived from casein representing the first: 2, 3, 5, 6, 7, 9, 11, 12,16, 23, 24 and 26 amino acids of αS1 casein stimulated the proliferationof dendritic cells, from 2.2% and up to 23% of total cells compared with0.1-0.2% dendritic cells in the cell samples incubated without Syntheticpeptides derived from casein (FIG. 7).

Synthetic peptides derived from casein stimulate Plasma cellproliferation in murine CFU-GEMM: The effect of Synthetic peptidesderived from αS1 casein on plasma cell proliferation in murine primarybone marrow cells was demonstrated under the same conditions outlinedfor the stimulation of megakaryocytes. Synthetic peptides derived fromcasein representing the first: 2, 3, 5, 7, 11, 16, 17, 18, 19, 20, 21,22, 23 and 24 and 26 amino acids of αS1 casein, significantly stimulatedthe proliferation of plasma cells, from 1.5% and up 12.3% of total cellcount, compared with 0.3% of total without Synthetic peptides derivedfrom casein (FIG. 7).

Synthetic peptides derived from casein stimulate Macrophageproliferation in CFU-GEMM: The effect of synthetic peptides derived fromαS1 casein on macrophage proliferation in murine primary bone marrowcells was demonstrated under the same conditions outlined for thestimulation of megakaryocytes. Incubation of cells with syntheticpeptides derived from casein representing the first: 7, 9, 16, and 23amino acids of αS1 casein significantly stimulated the proliferation ofmacrophages, from approximately 17% of total cell count in controls, tonearly 30% of total in cells incubated with Synthetic peptides derivedfrom casein (FIG. 7).

Synthetic peptides derived from casein stimulate Red Blood Cellsproliferation in CFU-GEMM: The effect of synthetic peptides derived fromαS1 casein on red blood cell proliferation in murine primary bone marrowcells was demonstrated under the same conditions outlined for thestimulation of megakaryocytes. Incubation of cells with Syntheticpeptides derived from casein representing the first 4 amino acids fromthe N terminus of αS1 casein (SEQ ID NO. 3) significantly stimulated theproliferation of red blood cells, from 53% of total cell count incontrols, to 71% of total in cells incubated with the synthetic peptidederived from casein (FIG. 7).

Synthetic peptides derived from casein stimulate Polymorphonuclear (PMN)cell proliferation in CFU-GEMM: The effect of Synthetic peptides derivedfrom casein on the proliferation of polymorphonuclear (PMN) cells inmurine primary bone marrow cells was demonstrated under the sameconditions outlined for the stimulation of megakaryocytes. Incubation ofcells with Synthetic peptides derived from casein representing thefirst: 3, 6, 7, 9, 16 and more, up to and including 26 amino acids ofαS1 casein significantly stimulated the proliferation of PMNs, from 1.6%of total cell count in unincubated controls, to between 2.9% and 14.9%of total in cells incubated with Synthetic peptides derived from casein(FIG. 7).

Synthetic peptides derived from αS1-casein stimulate Granulopoietic (GM)cell proliferation in CFU-GM: As mentioned hereinabove, formation andexpansion of CFU-GM (Granulocyte and Macrophage) colonies, and CFU-GEMM(Granulocyte, Erythroid, Macrophage and Megakaryocyte) coloniesconstitute one of the early events in the differentiation ofhematopoietic progenitor cells in the bone marrow. The effect ofsynthetic peptides derived from αS1 casein on the proliferation ofgranulocytes and macrophages in murine primary bone marrow cells wasdemonstrated under the same conditions outlined for the stimulation ofmegakaryocytes, with the addition of cytokine IL-3 and granulocytecolony stimulating factor (G-CSF). Incubation of murine bone marrowprogenitor cells with synthetic peptides derived from αS1 caseinrepresenting amino acids 1-22 (J, SEQ ID No. 21) and 1-6 (30-4, SEQ IDNo. 5), alone or in combination (FIG. 19) significantly stimulated theproliferation of granulocytes, when added along with G-CSF (18% and 25%increase for “30-4” and “J”, respectively, in the presence of G-CSF)(FIG. 19).

A similar effect of synthetic peptides derived from αS1 casein wasobserved on the proliferation of granulocytes and macrophages from humanbone marrow progenitor cells. Surprisingly, administration of syntheticpeptides derived from α-casein (“J”, SEQ ID NO; 21) enhanced thegranulopoietic stimulating effects of G-CSF by >50% (100 μg “J”). (FIG.20). Thus, synthetic peptides derived from αS1 casein or combinationsthereof are effective in augmenting the effect of granulopoietic factorssuch as G-CSF on bone marrow hematopoietic progenitor celldifferentiation and expansion.

Peptides derived from natural casein stimulate hematopoiesis in vivofollowing irradiation and bone marrow transplant: Myeloablative therapymay lead to life-threatening reduction in thrombocytes and leukocytes,which may persist despite administration of blood cells and growthfactors. The following demonstrates the effect of peptides derived fromnatural casein following irradiation and bone marrow transplantation.

Peptides derived from natural casein enhance leukocyte and plateletreconstitution following syngeneic bone marrow transplantation in mice:When sub-lethally irradiated (600 cGy), minimally bonemarrow-reconstituted, BALB/c mice (n=12) received 1 mg per mousepeptides derived from natural casein via intravenous injection one dayafter bone marrow cell reconstitution, significant increases inperipheral white blood cell counts on days 4, 6 and 15 post-treatmentwere noted, compared to controls receiving human serum albumin (FIG. 8),Platelet counts in the peripheral blood of both the treated and controlirradiated, bone marrow transplanted mice were equally depressed up to 8days post treatment. However, by the thirteenth day a clear advantagewas noted for the mice treated with the peptides derived from naturalcasein, demonstrating a significant increase over the human serumalbumin-treated controls which became even more pronounced by day 15(FIG. 9). Thus, peptides derived from natural casein enhance plateletand leukocyte reconstitution following transplantation with limitingnumbers of bone marrow cells. It is expected that this effect will befurther increased in reconstitution with optimal, rather than limitingnumbers of bone marrow cells.

Further, in another series of similar experiments, it was observed thata partially purified (diafiltration with a 1 kDa cutoff membrane)preparation of peptides derived from natural casein, comprising peptidesderived from natural αS1- and β-casein, significantly enhanced plateletreconstitution (by approx 25% over controls) in irradiated, bone marrowtransplanted mice.

Synthetic peptides derived from casein enhance leukocyte reconstitutionfollowing syngeneic bone marrow transplantation in mice: Whensub-lethally irradiated (600 cGy), minimally bone marrow-reconstituted,BALB/c mice (n=5 per synthetic peptide, n=10 in the control group)received 1 mg per mouse synthetic peptides (13-26 amino acids in length,see Table 3) derived from casein via an intraperitoneal injection oneday after bone marrow transplantation, a clear enhancement of leukocytereconstitution was observed. Significant increases in peripheral whiteblood cell counts over a 10 to 14 day period were noted with peptideshaving 15 (day 10: 1.72×10⁶ cells per ml; day 12: 6.54×10⁶ cells per ml)and 22 (day 10: 2.74 cells×10⁶ per ml; day 12: 5.20×10⁶ cells per ml)amino acids (see Table 3), compared to controls receiving human serumalbumin (day 10: 1.67×10⁶ cells per ml; day 12: 4.64×10⁶ cells per ml).Thus, synthetic peptides derived from casein enhance leukocytereconstitution following transplantation with limiting numbers of bonemarrow cells.

In a series of similar experiments, F1 mice (n=5 mice per group) whichhad been sub-lethally irradiated (750 cGy) andbone-marrow-reconstituted, as described above, received intravenousadministration of 1 mg of synthetic peptides derived from αS1-, orpeptides derived from natural αS1- or κ-casein, one day followingreconstitution. Peripheral white blood cell counts (FIG. 23) clearlydemonstrate the strong stimulation of early leukocyte reconstitution (5and 7 days post-transplantation) with both peptides derived from naturalαS1- and κ-casein, and synthetic peptides derived from αS1 casein.

Synthetic peptides derived from casein enhance platelet reconstitutionfollowing syngeneic bone marrow transplantation in mice: In order toconfirm the observed ability of synthetic peptides derived from caseinto enhance megakaryocyte proliferation in hematopoietic stem cellcultures (see FIGS. 6 and 7), the peptides' effects on plateletreconstitution in vivo was investigated. When lethally irradiated (800cGy), minimally bone marrow-reconstituted, mice (n=5 per group) received1.0 mg per mouse synthetic peptides 4P and 3a (6 and 12 amino acids inlength, respectively—see Table 3) in 4 daily intraperitoneal injections(4-7 days post-transplantation), a clear enhancement of plateletreconstitution over untreated controls was observed. Significantincreases in platelet counts at 10 and 12 days post transplantation werenoted for both peptides. Treatment with peptide 4P increased counts by29% (872×10³/ml compared with 676×10³/ml in the control group) at 12days post transplantation while treatment with peptide 3a increasedcounts by up to 35.5% (229×10³/ml compared with 169×10³/ml in thecontrol group) at 10 days, and up to 13.5% (622×10³/ml compared with461×10³/ml in the control group) at 12 days post transplantation. Thus,the same synthetic peptides derived from casein enhance megakaryocyteproliferation in vitro and platelet reconstitution following bone marrowtransplantation in vivo.

In an additional series of similar experiments, F1 mice sub-lethallyirradiated (750 cGy) and minimally-bone marrow reconstituted (3×10⁶cells) which received intravenous administration of 1 mg of syntheticpeptides derived from casein demonstrated a significant increase inplatelet counts. Mice receiving a synthetic peptide representing aminoacids 1-22 of αS1-casein had enhanced platelet counts of 34% greaterthan those of untreated control mice at 10 days post-transplantation(FIG. 22).

Peptides Derived from Natural Casein Inhibit In Vitro Infection ofLymphocytic T Cell Lines by HIV-1 Virus

Penetration of peptides derived from natural casein into lymphocytic Tcells: In order to investigate the mechanisms of immune stimulatory andanti-viral effects of peptides derived from natural casein, susceptibleSup-T1 and CEM cultured human T-cells were treated with peptides derivedfrom natural casein prior to in vitro infection with HIV-1 virus.Fluorescent microscopy revealed that FITC-conjugated peptides derivedfrom natural casein (100 μg per ml) penetrated the Sup-T1 cells whenincubated therewith as described above (FIGS. 10 a-f). A small amount oflabel was observed in the cytoplasm of the cells after 15 minutes (FIGS.10 a-b). At 30 minutes (FIGS. 10 c-d) more label was observed in thecytoplasm, with limited nuclear uptake. From 1-hour incubation and on(FIGS. 10 e-f), FITC-labeled peptides derived from natural casein wereobserved in the cytoplasm, but mostly they were concentrated in the cellnucleus. Analysis of the Sup-T1 cells by flow cytometry confirmedincreasing uptake of the labeled peptides derived from natural caseinfrom 5 minutes post incubation.

Peptides derived from natural casein enhance human lymphocyteproliferation: The presence of peptides derived from natural casein inthe culture medium resulted in increased Sup-T1 cell counts over aperiod of 14 days. The greatest increases in cell number at 7 days wasobserved for 50 μg per ml peptides derived from natural casein (42%),for 1000 μg at 10 days (30%) and for 600 μg (32%) at 14 days incubation(data not shown). Measurement of [³H]-thymidine incorporation by thecultured cells, providing a proliferation index, reflected the increasein cell number, with the most significant effect noted for 600 μg per mlpeptides derived from natural casein on day 10 and 50 μg per ml on day14 (FIG. 11). The reduced proliferation indices at 14 days probablyreflect cell overgrowth and nutrient depletion.

Synthetic peptides derived from casein enhance human lymphocyteproliferation: The presence of synthetic peptides derived from casein(all peptides listed in Table 3) in the culture medium resulted inincreased Sup-T1 cell counts over a period of 10 days. The increase wassimilar for all synthetic peptides. The greatest increases in lymphocytecell number in infected cells were observed for 250 μg and 500 μg per mlof peptide representing the first 9 amino acids (80% and 33%,respectively) (data not shown).

Peptides derived from natural casein Inhibit HIV-1 infection in humanlymphocyte cells: Susceptible CEM lymphocyte cells pretreated withpeptides derived from natural casein (50-1000 μg per ml) 24 or 48 hoursprior to incubation with HIV-1, or exposed to HIV-1 pretreated 3 hourswith peptides from natural casein, exhibited enhanced cell proliferationand reduced levels of viral infection compared to untreated controls.Cell counts and HIV-1 P²⁴ antigen assay at 15 days post infectionrevealed 100% inhibition of viral infection after 3 hours incubation ofviruses with 600-1000 μg per ml peptides derived from natural casein and98% and 99% inhibition after 24 hours incubation of cells with 50 and600 μg per ml peptides, respectively (comparing cell numbers withuninfected controls UIF). Longer incubation times were not found to bemore effective (FIG. 12). Although increasing concentrations of peptidesderived from natural casein enhanced cell proliferation at 3 and 24hours post infection, viral infection is most significantly inhibited inthese fastest growing cultures. An even more dramatic enhancement ofcell proliferation and inhibition of HIV-1 infection was observed inSup-T1 cells pretreated with peptides derived from natural casein beforeHIV-1 infection (average inhibition of viral infection of 96.7%, 88.7%and 95.7% for 3 hours pretreatment of virus, and 24 hours and 48 hourspretreatment of cells, respectively) (not shown). Thus, peptides derivedfrom natural casein penetrate human cultured lymphocyte cells and theirnuclei, enhance cell growth, and significantly reduce the susceptibilityof CD4 cells to HIV-1 infection. As such, peptides derived from naturalcasein are expected to be useful both at preventing HIV infection andfor post infection treatment of HIV infected and AIDS patients.

Synthetic peptides derived from casein inhibit HIV-1 infection in humanlymphocyte cells: The ability of synthetic peptides derived from caseinto inhibit HIV-1 infection in human lymphocyte cells was demonstratedusing CEM-lymphocyte cells under the same conditions outlined above.Susceptible CEM lymphocyte cells exposed to HIV-1 pretreated 3 hourswith synthetic peptides from αS1-casein (50-1000 μg per ml) exhibitedenhanced cell proliferation and reduced levels of viral infectioncompared to untreated controls. Further, 24 or 48 hours incubation withsynthetic peptides representing the first 3 amino acids of αS1 caseinconferred a significant degree of resistance to infection followingincubation with HIV-1. Lymphocyte cell numbers were 1.29×10⁶ (100 μg perml) and 2.01×10⁶ (500 μg per ml) in the treated cells as compared to theinfected HIV-1 control of 1.06×10⁶ (FIG. 13). HIV-1 infection levels inthe same cells, measured by the HIV-P²⁴ antigen assay at 7 days postinfection, was significantly reduced in the peptide treated cells (0.17and 0.14 ng P²⁴ Antigen/ml with 100 μg/ml and 500 μg/ml respectively),as compared to the untreated controls (0.52 ng P²⁴ Ag/ml).

Likewise, significant inhibition of HIV-1 infection was observed in theCEM cells exposed to viruses that had been pre-treated (3 hours) withthe synthetic casein derived peptide representing the first 5 aminoacids of αS1 casein.

Cell counts in the cultures incubated with 10 and 25 μg peptide 3P perml were 1.17×10⁶ and 1.26×10⁶ respectively, as compared to the infectedHIV-1 control of 1.06×10⁶.

HIV-P²⁴ antigen assay at 7 days post infection, revealed significantreduction in HIV-1 infection levels in treated cultures (0.26 and 0.18ng P²⁴ Ag per ml for 10 and 25 μg per ml respectively, as compared tothe control of 0.52 ng P²⁴ Ag per ml).

Likewise, 3 hours preincubation of the virus with the synthetic peptidederived from casein 4P, representing the first 6 amino acids of αS1casein had a significant effect on the susceptibility of CEM lymphocytecells to infection with HIV-1.

Cell numbers were most affected at concentrations of 25 and 250 μg perml (1.26×10⁶, and 1.59×10⁶ respectively, as compared to the infectedcontrol value of 1.06×10⁶).

Assay of HIV-P²⁴ antigen at 7 days post infection, revealed a dosedependent reduction in viral particles as compared to the untreated,infected control cultures (FIG. 13). Thus, the protection from HIV-1infection afforded lymphocyte cells by the peptides derived from naturalcasein is retained in synthetic peptides derived from caseinrepresenting as few as the first five N-terminal amino acids of αS-1casein.

Peptides derived from natural casein prevent development of glucosuriain Non-Obese Diabetic (NOD) mice: Non-Obese Diabetic (NOD) micespontaneously develop Juvenile (Type I, IDDM) Diabetes, an autoimmunecondition causing inflammation of the pancreatic β cells and ending indisease and death. Female NOD mice are extremely susceptible,demonstrating evidence of macrophage invasion of the pancreatic isletinterstitial matrix as early as 5 weeks old. A once or twice weeklyinjection of 100 μg peptides derived from natural casein for 5 weeks (5or 10 injections total) were completely effective in preventing theglucosuria associated with the onset and course of the disease. By 200days 100% of the untreated control mice (n=5) had become diabetic, andsubsequently died, while die treated mice (n=10) remained 100%euglycemic, all still surviving at 365 days (FIG. 14). Thus, peptidesderived from natural casein effectively protected geneticallysusceptible mice against the onset of this autoimmune inflammatorycondition.

Synthetic Peptides Derived from Casein Prevent Development of Glucosuriain Non-Obese Diabetic (NOD) Mice:

The preventative effect of synthetic peptides derived from casein on thedevelopment of glucosuria in NOD mice was demonstrated under the sameconditions outlined above, except that the mice were injected only onceweekly for five (5) weeks with 100 μg of Synthetic peptides derived fromcasein. The results of these experiments are presented in Table 4 below;

TABLE 4 The effect of synthetic peptides on IDDM in NOD mice PeptideIPGT TEST Derivative Urine 0 min. 60 min. code Healthy/Total* Sugar(preload) post load Y(SEQ ID NO:) 1/5 Negative 121 138 X(SEQ ID NO:) 3/5Negative 94 114 Megatlve 104 119 Negative 141 114 1a(SEQ ID NO:) 1/5Negative 88 106 2a(SEQ ID NO:) 4/5 Negative 215 183 Negative 112 119Negative 95 107 Negative 159 204 3a(SEQ ID NO:) 3/5 Negative 135 137Negative 205 197 Negative 201 211 A(SEQ ID NO:) 2/5 Negative 134 164Negative 105 107 B(SEQ ID NO:) 2/5 Negative 130 117 Negative 130 97D(SEQ ID NO:) 2/5 Negative 99 108 Negative 130 136 I(SEQ ID NO:) 2/5Negative 324 not tested Negative 124 138 J(SEQ ID NO:) 3/5 Negative 166not tested Negative 193 not tested Negative 186 not tested K(SEQ ID NO:)2/5 Negative 116 143 Negative 443 not tested Chay-13 2/5 Negative 123130 Negative 111 111 Chay-13 2/5 Negative 128 116 Negative 113 125Control 0/5 Blood was drawn from the paraorbital plexus at 0 min and 60min after the intraperitoneal injection of glucoe 1 g/kg body weight.Plasma glucose levels were determined with a Glucose Analyzer 2 (BeckmanInstruments, Fullerton, CA) and expressed as mmol/L. *Healthy and well =Sugar not detected in urine. Glucosuria = >1000 mg/dL. IPGTT performedwith 6 healthy female control mice: 0 min- 110 mmol/L; 60 min- 106mmol/L blood glucose.

The synthetic peptides derived from casein representing the first 9 (X)(SEQ ID NO. 8), 11 (2a) (SEQ ID NO. 10) and 12 (3a) (SEQ ID NO. 11)amino acids and higher chain length of αS1 casein, were highly effectivein preventing the glucosuria associated with the onset and course of thedisease.

Effect of treatment with synthetic peptides derived from casein wasevaluated after 25 weeks. At that time, all 5 mice in the untreatedcontrol group (n=5) had become diabetic, as indicated by the presence offrank (>1000 mg/dl) glucosuria (Table 4).

No glucosuria was detected in three of the five (3/5) NOD mice treatedwith the synthetic peptide representing the first nine (9) amino acidsfrom the N terminal of αS1 casein. Of the group injected with thesynthetic peptide of eleven (11) amino acids from the N terminal of αS1casein, no glucosuria was detected in four out of five (4/5) of the NODmice

In the groups of peptide treated mice in which glucosuria was detected,the onset was generally significantly delayed (by 3-5 weeks) relative tothe onset of glucosuria in untreated controls (data not shown),indicating a clearly protective effect of the peptides even whenincomplete.

The protective effects of shorter synthetic peptides derived from caseinhave also been studied in NOD mice. In an additional series ofexperiments similar to the abovementioned, administration of peptidesrepresenting the first 3 (1P) and 4 (2P) N-terminal amino acids of αS1casein effectively prevented the onset of glucosuria in the treated mice(assayed at week 16), while the untreated controls had all becomediabetic (100% glucosuria) (data not shown).

The glucose tolerance (IPGT) test performed after 25 weeks with thehealthy and well NOD mice, of the group injected with the syntheticcasein derived peptide of the first 9 amino acids (SEQ ID NO. 8), showedno evidence of abnormal glucose metabolism (normal glycemic values pre-and 60 minutes post-glucose loading).

In the group treated with the synthetic peptide derived from caseinrepresenting the first 11 amino acids of the N-terminal of αS1 casein(2a) (SEQ ID NO. 10), resting plasma glucose levels were somewhatelevated in two of the five mice (215 and 159 mmol/L), and remainedmildly elevated at (183 and 204 mmol/L) 60 minutes post load, indicatingmild diabetic tendencies. The other two mice remained within normalglycemic range throughout the test (Table 4). In general, the normalresults of the IPGTT reflected the absence of glucosuria in the healthy,surviving peptide-treated mice (Table 4). Thus, synthetic peptidesrepresenting only a few amino acids from the N-terminal of αS1 casein,as well as peptides derived from native casein dramatically reduce thesusceptibility of genetically predisposed NOD mice to onset ofautoimmune diabetic disease.

Synthetic casein-derived peptides significantly reduce Total Cholesterolblood levels (TC), Low Density Lipoprotein (LDL) and High DensityLipoprotein (HDL): Intraperitoneal administration of Synthetic peptidesderived from casein caused a significant reduction in the blood lipid(HDL, LDL and TC) values in experimentally hypercholesterolemia mice.After one week of the atherogenic Thomas Hartroft diet, the bloodcholesterol levels of the mice had risen to the levels of 318 mg/dl.

One week post treatment with 1 mg synthetic peptides derived from caseinper mouse, the group treated with the Synthetic peptides derived fromcasein representing the first 5 (3P) (SEQ ID NO. 4) and 11 (2a) (SEQ IDNO. 10) amino acids of αS1 casein, had significantly reduced TC, HDL andLDL values, compared to those of the control group [TC: 308 and 279mg/dl respectively; HDL: 42.5 mg/dl and 41 mg/dl respectively and LDL:247 mg/dl and 221 mg/dl respectively as compared to 393 mg/dl (TC), 54.5mg/dl (HDL) and 326 mg/dl (LDL) in the diet-inducedhypercholesterol-/hyperlipidemic control group] (FIG. 15). Thus,synthetic peptides representing the first few N-terminal amino acids ofαS1 casein effectively reduced experimentally induced hyperlipidemia andhypercholesterolemia within 1 week after a single, intraperitonealadministration.

Clinical Trials with Peptides Derived from Natural Casein:

Patients received a series of one, two or three intramuscular injectionsof 50 mg peptides derived from natural casein each, divided into threedepots each treatment, as indicated.

Peptides derived from natural casein stimulates hematopoiesis in cancerpatients: The hematology profiles of six cancer patients who hadreceived or were receiving chemotherapy were examined before andfollowing administration of peptides derived from natural casein, asindicated. Special attention was paid to changes in the Platelet (PLT),Leukocyte (WBC), Erythrocyte (RBC) and Hemoglobin (HGB) values,representing thrombocytopoiesis, leukocytopoiesis, anderythrocytopoiesis, respectively.

G.T., (Female patient, Patient 1): Patient had ovarian cancer, undergonea hysterectomy followed by chemotherapy. She received two intramuscularinjections of peptides derived from natural casein at two and then twoand one half months post operation. No chemotherapy was administeredbetween the first and second administrations of peptides derived fromnatural casein. Blood tests from 6 days post first injection, 7, and 13days post second injection reflect a considerable increase in plateletand WBC components, as well as increased RBC (FIG. 16).

E.C., (Female patient, Patient 2): Patient underwent a radicalmastectomy for lobular carcinoma in 1983, and six years later sufferedfrom gastric metastases. Three days prior to commencement ofchemotherapy, she received one intramuscular injection (in three depots)of peptides derived from natural casein by injection, and a second 10days after the chemotherapy. Although the blood counts from 10 and 16days post chemotherapy indicated an attenuation of the depressedhematological profile usually encountered following chemotherapy, themost significant effects of peptides derived from natural casein werenoted 3 days after the first injection, prior to the chemotherapy (FIG.16).

E.S., (Female patient, Patient 3): Patient was suffering from widespreadmetastatic dissemination of a breast carcinoma first discovered in 1987.Two years later, she received a first intramuscular injection ofpeptides derived from natural casein, and a second 23 days later. Noadditional therapy was administered during this period. Blood testsindicate a strong enhancement of PLT seven days after the firsttreatment and a significant increase in RBC and WBC seven days after thesecond treatment (FIG. 16).

J.R., (Female patient, Patient 4): Patient's diagnosis is breast cancerwith bone metastases. She received one intramuscular injection ofpeptides derived from natural casein 8 days before commencingchemotherapy, and another, 14 days later. The most significant effect isclearly seen in the rapid return of WBC levels followingchemotherapy-induced depression (FIG. 16).

D.M., (Female patient, Patient 5): Patient suffering from hepatic cancerwith widespread metastatic dissemination. She received threeintramuscular injections of peptides derived from natural casein at 10,8 and 6 days before receiving chemotherapy. A second series ofinjections was initiated 10, 12 and 14 days following the chemotherapytreatment. Although a significant effect on the hematological profile isnoted following the first series of injections and prior to thechemotherapy, the most dramatic improvements are seen in the rapidreturn of depressed post-chemotherapy values to normalized cell countsfollowing the second series of peptides derived from natural caseininjections (FIG. 16).

Thus, administration of peptides derived from natural casein to cancerpatients results in improved hematological profiles, specificallyenhanced erythropoiesis, leukocytopoiesis and thrombocytopoiesis, and iscapable of moderating and shortening the duration ofchemotherapy-induced depression of blood components.

Peptides derived from natural casein stimulates thrombocytopoiesis intransplant recipients with resistant thrombocytopenia: Prolongedtransfusion-resistant thrombocytopenia with episodes of severe bleeding,may be a life threatening complication of bone marrow transplantation,especially where traditional therapies are ineffective. Two patientswith severe resistant thrombocytopenia were treated with peptidesderived from natural casein.

M-1 (Female patient): 32 year old patient suffering from Acute MyeloidLeukemia in complete remission, following autologous stem celltransplantation. She had experienced two life-threatening bleedingepisodes, involving pulmonary hemorrhage and a large obstructivehematoma in the soft palate. At more than 114 days post transplantation,platelet counts were refractive to rhIL-3, rhIL-6, intravenous gammaglobulin, and recombinant erythropoietin. Following two intra musculartreatments of 50 mg peptides derived from natural casein (each treatmentdivided into three depots), her condition improved immediately. Alongwith the rapid return of normal platelet counts (FIG. 17), her distallimb bleeding with exertion and patechyae subsided, she was able toresume walking, and returned to her home overseas with no complicationsor side effects.

M-2 (Male patient): 30 year old patient suffering from Acute MyeloidLeukemia in a second complete remission following autologous stem celltransplantation, exhibiting totally resistant platelet counts andmassive gastrointestinal bleeding episodes. He required dailytransfusions of packed cells, had developed hypoalbuminia, and failed torespond to extensive therapy with rhIL-3, rhIL-6 and gamma globulin.Following two intramuscular treatments, each of 50 mg peptides derivedfrom natural casein in three depots 86 days post transplantation, rapidplatelet reconstitution (FIG. 18) and gradual discontinuation of thebleeding was observed. No further treatment was required, and thepatient is presently completely asymptomatic with normal platelet count.

Thus, one course of two intramuscular injections of peptides derivedfrom natural casein at 0.7-1.0 mg per kg body weight, each divided intothree depots, was effective in rapidly reconstituting platelet countsand diminishing associated clinical symptoms in patients suffering fromprolonged, transfusion resistant thrombocytopenia with life-threateningbleeding episodes.

Peptides Derived from Natural Casein Decreases Triglycerides and TotalCholesterol in Familial Hyperlipidemia:

M.S. (Female patient): Patient is a 38 year old female with familyhistory of hyperlipidemia. Before treatment with peptides derived fromnatural casein, blood chemistry profile revealed elevated totalcholesterol (321 mg per dl), triglycerides (213 mg per dl; normal range45-185 mg per dl) and elevated LDL-cholesterol (236.4 mg per dl; normalrange 75-174 mg per dl). One month after a single administration of 50mg peptides derived from natural casein (in three intra muscular depots)the hyperlipidemia was stabilized: total cholesterol was reduced to 270mg per dl, triglycerides were 165 mg per dl and LDL-cholesterol was 201mg per dl, still higher than normal range but significantly reduced fromthe pretreatment value. No additional treatment was administered. Thus,treatment with peptides derived from natural casein is effective inrapidly bringing about a significant reduction in otherwise untreatedhyperlipidemia in humans.

Peptides Derived from Natural Casein Stimulate Normoglobinemia in a Caseof Occult Bleeding:

D. G. (Male patient): Patient is a 75 year old male suffering fromanemia and hypoglobinemia (depressed RBC, HGB, HCT, MCH and MCHC)associated with extensive occult bleeding. One month after receiving oneintramuscular injection of 50 mg peptides derived from natural casein(in three depots), a significant reduction of the anemia was observed.After two months, RBC approached normal values (4.32 instead of 3.44 Mper μl), HGB increased (11.3 instead of 8.9 g per dl) and HCT, MCH andMCHC all improved to nearly normal values, despite the persistence ofoccult bleeding. Thus, one injection of peptides derived from naturalcasein seemed capable of stimulating erythropoiesis and reducing anemiaassociated with blood loss in humans.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents, patent applicationsand sequences identified by an accession number, mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent, patent application or sequence was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention.

1-176. (canceled)
 177. A method of preventing or treating a metabolicdisease or condition selected from the group consisting of NIDDM, IDDM,hyperlipidemia, and hypercholesterolemia, the method comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a peptide derived from the N terminus portion of alpha-S1casein.
 178. The method of claim 177, wherein said peptide is a fragmentderived from the N terminus portion of alpha-S1 casein by fragmentationof alpha-S1 casein.
 179. The method of claim 177, wherein said peptidederived from the N terminus portion of alpha-S1 casein is a syntheticpeptide.
 180. The method of claim 177, wherein said peptide derived fromthe N terminus portion of alpha-S1 casein has a sequence as set forth inone of SEQ ID NOs: 1-25.
 181. The method of claim 177, wherein saidpeptide derived from the N terminus portion of alpha-S1 casein is apeptide derived from natural casein, in combination with peptidesderived from natural non-alpha-S1 casein. 182-236. (canceled)